UTSC

BIOB10H3
MIDTERM EXAM
STUDY GUIDE
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BIOB10 Notes
Lecture #1

- Cells are the fundamental units of life, they grow and repair, and all living organisms
have them
Cell biology
- The study of cells at the microscopic and molecular levels
➢ Properties of cells, the structures of their organelles, movement of
macromolecules, specialization (i.e differentiation), and interactions between cells
Discovery
- Robert Hooke (1665) cork cells under microscope
- Anton van Leeuwenhoek (1684) pond water, plaque
- Scheeiden, Schwann, and Virchow proposed the Cell Theory
1. All organisms are composed of 1 or more cells
2. The cell is the structural unit of life
3. Cells can only arise by division from a preexisting cell
Basic Properties of a Cell
1. Cells are highly complex and organized
2. Cells possess a genetic program and the means to use it (i.e genes proteins)
3. Cells are capable of producing more of themselves (i.e mitosis/meiosis)
4. Cells can acquire and utilize energy (i.e photosynthesis/respiration)
5. Cells carry out chemical reaction (i.e metabolism) and use enzymes that aid in these
metabolic processes
6. Cells engaging in mechanical activities
➢ Transporting materials 1. Intracellular transport (w/I cell), 2. Intercellular
transport (between cells)
➢ Assembling and disassembling of structural (done by the cytoskeletal elements)
➢ Whole cells move
7. Cells respond to stimuli
➢ Cell surface proteins receptors they have ligands bind to the receptors,
initiating a response
➢ Cells are very responsive to cues/signals that help them interact with their
environment
8. Cells can self-regulate (i.e DNA repair enzymes, determining when to stop/start dividing)
9. Cells can evolve genetic changes that can lead to cellular changes, that are beneficial
and retained in their future generations
- Pasteur experiment found that life does not arise spontaneously (eg. Swan neck
flask experiment)

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Types of Cells
1. Prokaryotic cell
a. Nucleoid – genetic material not
bounded by a membrane (scattered
in 1 region of the cell)
b. Simpler structures
c. Less DNA that eukaryotes
typically have a singly, circular
chromosome
d. Undergo Binary Fission

2. Eukaryotic Cells
a. Membrane bound
organelles (i.e nuclear membrane)
b. Structurally more complex
complex cytoskeletal system
(involve different pathways in the
cell)
c. More DNA than prokaryotes typically several chromosomes composed of single,
linear DNA molecules
d. Divide through mitosis/meiosis
Similarities Between the Two Cell Types
- Genetic codes are identical information is always encoded in the DNA
- Share metabolic pathways (i.e synthesis of ATP)
- Shared structural elements (i.e the cell membrane)
Origins
- Prokaryotic cells came before eukaryotic cells fossil record
➢ See similarities between eukaryotes and prokaryotes including genetic code and
metabolisms
- Endosymbiont theory: a combination of 2 cells living together in a symbiotic
relationship; one cell
li es i side the other
cell
➢ Both cells benefit
from each other (but
one lives inside the
other)
-

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Types of Prokaryotes
1. Archaea (archaebacteria) extremophiles
2. Bacteria (eubacteria) all other bacteria
➢ The cyanobacteria being the most complex (with an complex internal organization of
their membrane)
➢ Archaea are actually closer to Euk. Than eubacteria
Types Of Eukaryotes
1. Unicellular protists, ciliates, flagellates
➢ Everything they need to do is done by this one cell
2. Multicellular humans
➢ Different activities being carried out by different types of specialized cells cell
differentiation
- Oocytes need 250 cell differentiation events to occur to create functioning human
Cell Differentiation
- The process by which an unspecialized cell becomes a specialized any cell can
become specialized
- Differentiation primarily occurs due to cues fro the cell’s e viro e t the env. Of
the cell dictates what type of cell it will become
- The types of signal received by the cell, depends on the location of the cell within the
embryo
➢ changes in morphology (appearance)
➢ cells expressi g cell-specific ge es unique proteins that come from expressed
DNA that will help that specific cell
• house-keeping proteins (eg. For metabolism) remain the same, just as in other
cells
➢ organelles stay the same, but their number and location may differ (i.e RBC eject
nucleus upon maturity)
- Units used are: Angstrom (10e-7), Nanometer (10e-6), and Micrometer/Micron (10e-3)
Cell Culture
- Cells are gro outside of the ody’s atural e iro e t, i a sy theti , o trolled
env. Called in vitro
- Cells are grown in plastic flasks filled with defined media the place for cell growth is
the medium
- Primary culture is obtained directly from the organism
➢ Mostly embryonic tissue and can divide 50-100x
- Cell line: primary cultures that have undergone genetic modifications to allow them to
grow indefinitely in culture (i.e derived from primary culture and modified to grow
indef. On plastic)
➢ Can occur spontaneously (transformed mouse cells) or tumour tissue can be used
(HeLa cells)

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- 3-D Cell Culture

➢ uses pseudo env. Of what cells exp.
In the body ECM (created in artificial
flasks w/ ECM and find that the cells
actually come together and create small
tissues)
• in essence, changed media, changes
cell behavior
• more complex organization occurs in
3-D culture, and cells respond faster to changes compared to on a 2-D matrix
Advantages of Cultured Cells
- cells can be obtained in large quantities
- most cultures only constitute only one type of cell (monoculture)
- important cell biological phenomena can be studied using
cultured cells (i.e cell movement/division)
- cells can be induced into differentiating in culture
- cultured cells can also be used to test the activity of drugs
(i.e cell line has HGH used on it
➢ use hormones or growth factors
Studying Cells
• Brightfield light microscopy
➢ Produce enlarged images of an
object
➢ Cells are placed on a stage
between the light source and our eyes
(ocular lens)
➢ View light that is diffracted by the cells
- Preparation:
1. Fix cells (fixation) and stop materials from moving
2. Stain cells (dye makes their structure more visible)
3. Mount on slides to view
- Get tissues, slice (embed and section), then place on slides
- Stains used: Haemotoxylin and Eosin staining (H&E)
Haemotoxylin stains nucleic acids blue and Eosin stains proteins
pink
➢ i.e neuron for CJD brain holes represent areas with
neither pink OR blue stain
• Other light microscopes include: Phase contrast and
Different Interference Contrast (DIC)
o To see details of unstained specimens like living cells
o Use spe ialized opti s to i rease o trast of these
specimens
o Darker= sharper change in refractive index (contrast b/c all organelles have
diff. refractive indexes)

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Use of Light Microscopy:

- Examine cellular structures (fixed or alive) are relatively high resolution
- Resolution: the extent to which the detail of a specimen is retained in the image
➢ Dependent on the wavelength of light
• Electron Microscopy (EM)
➢ Uses ele tro s are light sour e
have short wavelength
➢ Image is formed when e- pass
through a specimen much better
resolution
➢ Resolution of a microscope is
inversely proportional to the
wavelength of its light source � ∝
1/�
o The longer the wavelength, the
poorer the resolution
• Transmission electron microscope
(TEM)
➢ Specimens are fixed, embedded, and
sectioned stained w/ heavy metal
solutions (that bind to cellular
macromolecules)
o Metals scatter e- and the image of
scattered elections is caught on
photographic emulsion
✓ The dark portions of the
image are the regions where the
electrons have been scattered away
by metal atoms i.e nucleus

deflects e-, and appears

very dark
• Cryo electron microscopy
➢ Gets sample that features all the
states the protein can be in
constructs the average of those
states and then creates a model
➢ Cell is fixed by immersing into
something very cold flash freeze
(cryofixate cell)

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• Freeze fracture and freeze etching

➢ Look at things in membrane or w/I boundaries
➢ Cells are rapidly frozen in low. Temp liquids knife strikes cells creating a fracture
plane that spreads from point of initial contact and splits the cells in 2
o Cellular structures either deviate upward/downward of irregular surface such
that one side features the portion, while other side shows the outline of the
structure
➢ Hea y etals are pla ed o top of fra tured surfa e to get a repli a old of
plane carbon is
deposited over the
metals to cement them
in place and is then
viewed under EM

• Scanning Electron Microscope (SEM)

➢ Specimens are prepared by carefully drying them after drying, they are coated w/
carbon and metals (allow an e- dense component that helps disperse e-)
➢ Image is formed by electrons reflected back by the specimen (referred to as
a ks attered ele tro s (looks at bounced e- and get info)
➢ Provides a surface view of cells/tissues/ and whole animals
o Used for how diff. cells types come together and make organisms
➢ Best for looking at surface projections that might affect other cells

Cell Replacement Therapy

- Bone marrow transplants destroy all blood cells and then donor bone marrow cells are
transplanted into the blood stream
➢ Hematopoeitc stem cells (HSC) are in bone marrow that give rise to all blood cells
Stem Cells
- Undifferentiated cells that can self-renew and differentiate into 2 or more mature cell
types
- Most organs contain stem cells adult stem cells (help them produce new cells)
- Ca ’t e ultured for lo g a d differentiation capacity is limited

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Human Embryonic Stem Cells (ES)

- Derived from human embryos (IVF clinics)
- Are pluripotent (can differentiate into any type of cells)
- Can be cultured for extended periods
➢ Good for cell replacement therapy eg. ES-derived oligodendrocytes (for spinal
cord injuries)
- Problems: immunologic reaction (body may not recognize cells from donor)
Customizing ES Cells
Oocyte has - Involve changing the genetic
Usually skin patient
makeup of the cells to match that of the
cell DNA
patient who needs the cells transplant
- Problems: creating an embryo just
as a source of ES cells is unethical
Induced Pluripotent stem cells (iPS)
➢ reprogrammed somatic cells by
Remove genetic introduction of specific genes into them
info of oocyte
(remove the need for human oocytes)
1. take adult cells
2. add seq. of DNA that will create a
type of induced stem cell
3. give cell growth factors/hormones
that create a specific cell

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Trans-differentiation
- take something that is already differentiated and make it a different type of its cell (i.e
convert � ����
- cells make insulin and are lost in type 1 diabetes knowledge of the expression of 3
genes that allow beta differentiation is important
- use virus to deliver 3
genes to alpha cells
causes beta
differentiation and
insulin production

Tree of life
- new study finds that
some archaea bacteria
have ancient form of
Eukaryotic DNA

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LECTURE #2

Biological Macromolecules
- long chains of carbon atoms used to construct bio. Molecules (linear, branched, or
cyclic)
➢ simplest are hydrocarbons
➢ hydrogen is often replaced by functional groups
- functional groups: particular atom groupings that behave as a unit affect properties
and chemical reactivity

Functional groups
- affect the properties of biological molecules
b/c: contain e- negative atoms (N,O,P,S),
have polarity/reactivity, provide a charge
when ionized

Formation of Macromolecules
- formed by polymers of building blocks known as monomers formed through
condensation reactions (remove water)
➢ broken down in hydrolysis reactions (add water)
Types of Biological Macromolecules
1. Carbohydrates
- Formula: CH2O
- Sugars usually have 3-7 carbons ea h sugar’s ar o s are li ked to hydroxyl groups,
except the one that has a carbonyl group (C=O)
- Internal carbonyl position = ketose
- Carbonyl at one end aldehyde = aldose sugar
- A sugar w/ 5+ carbons form closed rings glucose is a 6-membered ring
- OH of Carbon 1 above = �, OH of Carbon 1 below =

- Covalent linkages between C1 and hydroxyl of other sugar

is how linkages form C-O-C linkage between sugars (1-4)
➢ Types of Carbohydrates
1. Glycogen (store chemical energy), (alpha 1-4 bonds)
2. Starch (store chemical energy in plants) (alpha 1-4)
3. Cellulose (structural polymer) Beta 1-4
4. Chitin (polymer of N-acteylglucosamine) structural
polymer of exoskeletons Beta 1-4

2. Lipids
- Large group of nonpolar biological macromolecules
- Mainly C,H, and O dissolve in organic solvents but not in water
- Lipids with important functions: Fats (storage), Steroids (messengers), Phospholipids
(bilayers and membranes)
- Fats = triacylglycerol = glycerol + 3 fatty acids
➢ Fatty acids: long hydrocarbon chains with one single carboxyl at one end
o Vary in length
✓ No double bonds = saturated
✓ Double bonds = unsaturated (usually plants)
- Fats 3 fatty acid chains linked to glycerol through ester bonds
- Steroids: complex ring structures (4 hydrocarbon rings) i.e cholesterol
➢ Not present in plants
- Phospholipids: glycerol + 2 fatty acids + phosphate group
➢ Major component of plasma and organelle membranes
➢ Amphipathic
➢ Positively charged choline group attached to phosphate = phosphatidyl choline
➢ arrange into bilayer

3. Nucleic Acids

- DNA and RNA (tRNA, rRNA, mRNA)

- DNA is expressed in subsets when it comes to diff.
cells
- Composed of: 5 carbon sugar, phosphate group
li ked to 5’ ar o a d a itroge ous ase
 ➢ ribose sugar has OH, instead of H

- nucleotides are joined by sugar-phosphate linkages 3’ h dro l atta hes to 5’

phosphate = phosphodiester bond
➢ read 5’-3’ / 5’ phosphate is free o top a d 3’ h dro l is free at the otto
- Nitrogenous bases Purines (single ring) Adenine, Guanine, Pyramidine (double)
Cytosine, Thymine/Uracil
- Hydrogen bonds hold together the 2 strands of DNA in a double helix and the strands
run antiparallel
➢ C-G bond = 3 H-bonds // A-T bond = 2 H-bonds
- Helical shape forms b/c bases want to stack (hydrophobic) and P is doesn’t want to
interact w/ other P

4. Proteins
- About 10,000 known proteins and carry out almost every cellular function
- Composed of amino acids H,C, O, N, and also S or P
- Amino acids have: Amino terminal (NH2) and Carboxyl terminal (COOH) both are
separated by an alpha carbon
➢ Side chains give them their variability
R groups of Amino Acids
- 4 categories: Polar Charged, Polar Uncharged, Nonpolar, Other
1. Polar Charged – can form ionic interactions
➢ Arginine (+) in histones bind –ve DNA and package DNA
2. Polar Uncharged
- can form H-bonds

3. Non-polar: mostly hydrocarbons, hydrophobic, usually in the core of protein

structures (away from water) involved in membranes and interact w/ lipid bilayer

4. Others
 ➢ Have symmetry on how they interact
Can bond w/ other cysteine
to create disulphide When in long
linkages chain, won’t
be able to H-
bond

➢ cysteines residues can form disulphide bonds with

each other under oxidizing conditions
o Oxidizing env. ER
o Reducing env. Cytosol

Protein Structures
- Extracellular proteins are made in the ER
- Proteins are read in the N (+) C (-) direction
1. Primary Structure: specific sequence of amino
acids and are determined by the dequence of the gene
encoding the protein
- 20n variations of proteins (n= # of proteins of AA in
the chain)
- determines the 3-D structure, shape, and function
of the protein
➢ changes can have big consequences sickle cell anemia

- Christian Anfinsen: discovered that

the linear sequence of AA in a protein
contains all the info for it to fold
properly
➢ Found that unfolded
ribonuclease refolded by itself
Steps in Protein Folding
- Protein is synthesized and the
folding of domains of the protein
begin right away (form h-bonds, is co-
translational)
- Interactions between neighboring
AA leads to initial folding 2*
structure
- Once structure has formed, remainder of folding is done by hydrophobic interactions
that force non-polar AA into the core of the protein hydrophobic protein core
 - The protein may take several routes to folding each have final same folded state
Chaperones
- Help proteins that aid in the folding of newly made proteins by preventing
inappropriate interactions
➢ H dropho i i tera tio s a happe whe protei has ’t fully folded
chaperones help protect these sites and help proteins properly fold
- Also prevent inappropriate interaction w/ other cellular components
- Bind hydrophobic segments of proteins
- Can be single proteins or a large, protein complex with intricate structure
- Located in the cytosol of mammalian cells (i.e Hsp70 or TriC)
2. Secondary Structure
- Conformation of portions of the polypeptide chain and are arranged to maximize the
number of H-bonds made between neighboring AA (of backbone only)
- Has 2 Types of Folding Patterns: − � � �� − �� �
➢ Protein structure is dictated by the primary sequence
Alpha Helix:
- Hydrogen bonding that links the
C=O of one to the N-H of another
(every 4th amino acid)
➢ Creates cylindrical twist and
the R-groups project
outwards
o Have peptide
backbone interactions
o Specific R-groups
dictate what forms an
alpha helix
Beta-Pleated sheets:
- H-bonds (from carbonyl and
amide groups) extend from one
part of the chain to another and
the polypeptide segments lie
side by side (not necessarily 4 AA
away)
 ➢ Have antiparallel (preferred)
and parallel orientations
• proli e a ’t ake H-bonds
(R group binds to own made
group)
➢ good for antiparallel beta
sheet
- 2* structure can have: alpha
helices, beta sheets and loops
and turns

Adjacent sequences
form H-bonds

3. Tertiary Structure
- Conformation of the entire protein
- Interaction between R-groups
➢ Hydrophobic interactions, Van der Waals interactions (charged, non-polar R-groups
used), di-sulphide bridges (cysteines, forms more organization)
➢ 2 types: Fibrous and Globular proteins
- non-covalent bonds that hold protein
tertiary structure together
- Fibrous proteins: elongated shape,
structural (keratin, collagen),
- Form long strands or flattened sheets that
resist shearing force
- Globular proteins: compact shape
(hydrophobic residue is inside protein),
chain folds into complex shapes, and
proteins have functions w/I cell
(enzymes/hormones/most proteins)
- Despite 1* structure differences, related
tertiary structure relation shows proteins
might be form same part/serve similar
function (i.e actin/MreB)
 Bind actin Folding
- parts of protein folded into tertiary structure can have diff. fxns
4 different proteins comprise 4 diff. domains

from other regions (different regions fold independently, co-

translational)
➢ domains of a protein (different folding regions)
- can be swapped between proteins and allow for unique protein
activities (i.e binding to different molecules)

4. Quaternary Structure
- link multiple proteins to form large complexes with multiple
su u its multiprotein complexes
- Intermolecular interactions of R-groups disulfide bonds, non
covalent interactions
Bind Ca2+
➢ Come and create 1 full protein w/ a new complexity
Protein Modification and
Regulation
- Cleaved into smaller
polypeptides (proform in 2 proteins of
smaller segments) same seq. that
- Sugar chains come together to
(glycoproteins) fxn
- Lipids added (anchor cell
membrane)
- Metals/ions
- Phosphate groups [a
covalent modification) have 2 of 2 diff.
➢ Signaling/changes protein subunits
conformation or
interaction
- GTP or Ca2+ binding (alters protein activity)
- Degradation (control life span)
Regulating Proteins
- Regulate by degradation diff. proteins have diff. life cycles (i.e mitotic cyclins or
crystalline in eye)
➢ Controlled by degradation pathways
- Degradation pathways: lysosomes, autophagy, proteasomes (90% of the degradation)
 Proteasomal Degradation
- catalytic subunit protein is put through
this chamber
- autophag does ’t work is l soso es do ’t
- cell compensates for loss of one type of
regulator by increasing activity of another
- the proteasome degrades proteins for
turnover, but also degrades misfolded
proteins
➢ finds the proper protein to degrade through
ubiquitination tag
o ubiquitin (Ub) = 76 residue protein multiple copies of it are attached to a
protein targeted for Proteasomal degradation (polyubiqutination)
o requires activity of E1, E2, and E3 enzymes that work in sequence to bring
polyub.
- There are very few
E1s (work on many
proteins) and lots of
E3s (work on very
specific proteins)
• E1 activates Ub (ATP
to AMP+PP)
• E2 (conjugating
enzyme) removes E1
and has Ub transferred
onto it
• E3 (ligating enzyme)
takes Ub and adds it
onto substrate (while E2 is removed) marked w/ lysine side chains that are
• polyub.
 Binding
- Non-covalent binding to GTP can act as a switch for
regulating protein activity
GTP in,
➢ GTP Binding proteins = GTPases
GDP out
• GTP bound = active regulates activity of target proteins
in this state
• GDP bound = inactive
- GEFs and GAPs aids in regulating the switch that regulates
these proteins
➢ GAP makes hydrolyzing GTP faster removes Pi

Phosphorylation
- Most common covalent modification
that is used to regulate protein activity
- Reversible addition of a phosphate
(PO4) to the hydroxyl groups on side
chains of serine, threonine or tyrosine
- Kinases = add phosphate //
Phosphatase = removes phosphates
(??)
- Phosphorylation alters conformation:
➢ Allows for protein-protein
interactions
➢ Allow for altered intrinsic activity
(eg. Enzymes)
➢ Causes a change in the localization of the protein
• Causes large signaling pathways

CJD
- Creuzfeld Jacob Disease protein aggregates in the brain (fatal disease)
- caused by changes in the 2* structure of PrPc prion protein goes from alpha to strong beta
sheet
- PrPsc is misfolded caused neuronal aggregates (amyloid fibers or plaques)
- Protein only Hypothesis
> PrPsc is infectious can convert PrPc into a misfolded protein 1* seqeuence is identical, but 2*
is different
o Intermolecular interactions cause cause PrPsc to act as a chaperone and turns it into a
homodimer
✓ Homodimer then stacks on top of eachother creating amyloid fibers and tangles
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LECTURE #3

Organelles in Cells

- Keith R. Porter
lace like network that
spread throughout the
cytoplasm
- Endomembrane
system: ER, Golgi,
Lysosomes,
Endosomes, Vacuoles
Isotopes
- Radioactive
isotopes release
radioactivity when
they disintegrate
- Radioactive labelling of proteins
➢ Include radioactive AA w/ 1 or more radioactive atoms in a
growth media the proteins made in the cell will become
labeled with these radioactive AA
➢ Ribosomes insert these radio AA into proteins (like normal
ones)
➢ Synthesize AA w/ radioisotopes cell will make
radioactive proteins with radioactive amino acids
Autoradiography
- Energy emitted from radioactive
atoms used to label proteins can be
used to activate a radiation sensitive emulsion
- Silver grains are visible in the EM grains show the
location of the proteins in the cell
➢ Dark patches are where the proteins are
Endomembrane system Function Experiment
- Albert claude and Palade labelled proteins w/ radioactive
isotopes and detected them by autoradiography to
determine fxn of endomembrane system
• Experimental setup
- Pancreatic acinar cells (digestive enzymes) added radio
AA to medium radio AA incorporated into proteins
(digestive enzymes) fixed cells used autoradiography
and EM
➢ Silver grains showed where the proteins were synthesized
Pulse Chase Experiment

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- Cells were pulse-labeled (w/ radioisotopes) the ashed a d chased i ediu

containing NO radioisotopes
- Radio AA added to culture medium wash and replace after 3m chase the proteins
to find where they go after a specific time frame
- Found proteins move from ER Golgi out of cell
➢ Synthesized in ER Golgi vesicle secreted
Endoplasmic Reticulum
- The endomembrane system is also called the secretory pathway the ER is the
Ea ly sec eto y path ay o ga elle
- A system of membranes enclose a space (lumen) that is separated from the cytosol
luminal space
- ER has 2 sub compartments Smooth ER (SER) and Rough ER (RER): RER is studded w/
ribosomes, SER has no ribosomes
- SER: synthesize steroids, store calcium, detoxify alcohol
- RER: synthesize secretory pathway proteins (and some lipids), site of protein folding and
modification
Proteins made in the ER
- Synthesized on ribosomes attached to the RER
- Secreted proteins Hormones, neurotransmitters
- Transmembrane proteins Proteins on the PM
- Soluble proteins that reside in the endomembrane system organelles (eg. ER, golgi,
lysosomes, vacuolar proteins)
Proteins NOT made in the ER
- Synthesized on free ribosomes on the cytosol
- Cytosolic proteins many enzymes; cytoskeletal proteins
- Peripheral membrane proteins membrane-associated proteins
- Nuclear proteins
- Proteins that reside in peroxisomes, chloroplasts, and mitochondria (these proteins are
imported into the organelles)
- RER correlates w/ protein secretion
➢ i.e antibody secreting plasma cell has lots of RER, T-lymphocyte has very few
Synthesizing Proteins on Membrane-bound or Free ribosomes?
- Cell differentiates proteins for the ER/cytosol based on signal sequences that direct
their translation
➢ Signal sequence containing proteins are targeted to the RER once the signal is read
and emerges out of ribosome
✓ Ribosome moves away from cytosol and towards RER
- The signal sequence is a protein sequence translation begins on free ribosomes and
only after the signal is translated, does the whole complex (including the ribosome(
moves to the ER membrane
➢ i.e – all proteins are initially synthesized on the cytosol, then move to the RER once
the signal sequence is synthesized

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Signals and Protein Synthesis on membrane-bound ribosomes

1. signal recognition by the SRP after recognition
are recognized by the SRP
First few AA are rec. as sig. seq, and

and binding, translation stops

SRP mediated targeting of ribosome nascent
protein complex is moved to the ER membrane
SRP brought to the SRP receptor (allowing to come to
right location)
Docks free ribsome-protein complex at the RER
translocon = Sec61 complex (a protein channel that
move macromolecules from 1 side to another)
Docking of the ribosome at the ER membrane and
interaction of the nascent protein w/ the translocon
SRP leaves and sig. seq. now interacts w/ RER
membrane will resume translation
SRP is brought near the SRP receptor

a) Translocation (moving protein into the ER) of the

nascent protein into the ER lumen assisted by
chaperones such as Bip
translocation = movement into the endomembrane
system
b) Signal sequence removal by signal peptidase in the
ER lumen (signal peptidase removes signal from the
N-terminus and translocation continues)

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Synthesis of Transmembrane Proteins

The sig al se ue ce is clea ed a d t a slatio is happe
As translation happens, a hydrophobic stop-transfer signal is detected
that signal that it is not soluble and wants to be in the membrane
This signal is recognized, and this sequence is translated sideways
into the membrane
note: BiP is already folding other domains of the protein
Hydrophobic part of protein is now inside the membrane
Note: N terminal is inside, C is outside
Whether N is on inside or outside, depends on the charge of the
translocon
Cystolic end of translocon accommodates +ve charges (charge is –ve)
Luminal end accommodates –ve charges (charge is +ve)

Signal Sequence
- produced by Blobel and Sabatini
- p otei s co tai a se ue ce of AA at thei N-terminus that acts
as a sig al fo t a slocatio
- typically, 15-40 AA, hydrophobic (6-15 AA), and at N-terminus
SER Proteins?
- Proteins in SER correlate w/ fxn of the organelles
➢ Calcium binding proteins and channels
➢ Oxidizing enzymes that convert harmful substances into water soluble excretion
products
➢ Detoxification function
➢ Steroid hormone synthesize
- Proteins are targeted for SER: Cytosol RER SER signal

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Protein Folding in ER: Chaperones

- a multitude of proteins are chaperones: BiP, GRP94, Calnexin (CNX) [is an ER membrane
bound protein], Calreticulin
- chaperones are most abundant in the ER
- PDI (protein disulphide isomerase) assists in the formation of disulfide bonds and is also
therefore, required for the proper folding of proteins
➢ Some fold incorrectly, so PDI fixes those tries to find the best cysteines to form
their shape
Post-Translational Modifications in ER
- Most common modification of proteins is glycosylation (addition of oligosaccharides)
➢ Proteins modified with sugars are glycoproteins
- Sugars on proteins: help them fold, help them interact w/ other macromolecules
N-glycosylation in the ER
- Nearly all protein in the ER are modified co-translationally by glycosylation
- Enzymes that add sugars are called glycotransferases
- N-linked glycosylation: N = Asparagine sugar chains are added to asparagine in a
protein that is located in a motif
➢ Asn-X-Ser/Thr if this seq is present, sugar with be added to Asn
• N-linked glycosylation
occurs:
1. Sugars are built on a lipid
called dolichol on the cytosolic
face of the ER
2. Dolichol then flips over into
the luminal side and more sugars
are added
3. Sugars are then transferred
using glycotransferase onto the
protein

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ER Quality Control System

- Checks to make sure proteins are folded
properly
- Allows time for hard to fold proteins to
reach final state
- Destroys terminally misfolded proteins
• The Calnexin Cycle
1. 2 glucoses are trimmed by glucosidases I
and II from the glycoprotein
2. Monoglycosylated protein binds to
Calnexin which helps protein achieve its final
form
3. Glucosidase II trims final glucose from
protein that has interact w/ Calnexin trimming final glucose release
protein form calnexin
4. If improperly folded, UGGT adds another glucose
5. Re-glycosylated protein gets to try again and fold
6. If properly folded, leaves calnexin cycle and the ER
7. If terminally misfolded, is degraded by the proteasome
8. Protein to be degraded is moved from the ER to the cytosol where
proteasome degrades the defective protein this mechanism is used by
cells to prevent the accumulation of defective proteins
If Proteins continue to Misfold?
- Typically destroyed by proteasome in ERAD (ER-
associated degradation)
- If large amounts of unfolded/misfolded proteins
accumulate in the ER (i.e due to changes in
temp/cellular damaging agents), cells have evolved
the: Unfolded Protein Response
➢ During UPR: cells increase synthesis of chaperones
and proteasomes, decrease translation of new
proteins involved in the UPR
- Translation factor elf2� is phosphorylated, and a
change in gene expression happens causing the
increased expression chaperones/proteases to be
made
➢ If this still does not work, programmed cell death

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Cystic fibrosis
- Genetic mutation in gene that encodes the CFTR protein
➢ Normal protein: important in PM for allowing transport of Cl- ions that are required
for the hydration of airways and suspension of bacteria in the airways
✓ Non-functioning = unhydrated airways, build-up of mucus, biofilm (layers) of
bacteria
- For patients, phenylalanine is deleted at position 508 Δ508 (deletion at 508) and
results in the creation of a mutant CFTR that is degraded by ERAD Cl- is ’t pu ped
out, H2O ENTERS cell (feels like drowning on the inside)

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LECTURE #4

PROTEIN TRANSPORT THORUGH THE ENDOMEMBRANE SYSTEM

VESICLE BUDDING
- Vesicle budding involves:
➢ A donor compartment a vesicle will form here
➢ The vesicle a membrane closed dynamic cellular
organelle that is involved in the selective transport of
proteins/materials to other compartments, only selects
certain proteins and is formed & released as necessary
➢ A recipient compartment vesicle will fuse with this
compartment and deliver its contents
- Buddi g esi les ha e a coat of soluble (Cystolic)
proteins that assemble on the membranes of the donor
compartments

Coat Proteins
- allow the curving of the membrane to be a vesicle
- can mechanically force the membrane to curve and form a budding vesicle
- can bind to specific (Cytosolic end) transmembrane proteins receptors that in turn
bind to specific soluble proteins/components (cargo) to be recruited into the budding
vesicle
transmembrane receptor type of receptor
dictates what type of cargo, type of coat also
determines type of cargo
- vesicle coats were discovered in EM as a
fuzzy electron dense outer layer coated
buds pinch off into coated vesicles

Vesicles have Coats

- COP II vesicles: transport proteins forward from ER to the Golgi
➢ Anterograde transport
- COP I vesicles: tra sport protei s a k ards fro Golgi to the ER a d ithi the Golgi
from TGN to the CGN
➢ Retrograde transport
- Clathrin coated vesicles (most common): transport proteins to the lysosomes, vacuoles
➢ Involved in: protein sorting to lysosomes & vacuoles), transport of proteins from PM
to the endomembrane compartments, endocytosis
Vesicular Transport out of the ER

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- The COP II coated vesicles form at the ER membrane and fuse with the Golgi:
anterograde transport of proteins
- COP II coat = several proteins in a large complex
➢ One is a GTP binding protein Sar I (GTPase)
Other nucleotides
- GTP: guanosine triphosphate (provides a non-
covalent switch)
➢ Binds many proteins and regulates their
activities
➢ Can also be source of energy hydrolysis of
GDP and Pi
- Many GTP binding proteins exist in 2 states:
➢ GTP BOUND = active // GDP BOUND = inactive
Assembly of COP II Coats
1. an ER-membrane GEF recruits Sar I to the ER (interacts w/ SAR I w/ GEF adds a
phosphate to get a GTP change in structure diff. shape = diff. interactions that
increase the curvature of membrane) and catalyzes the exchange of the bound GDP for
GTP
2. this GDP to GTP switch (inactive to active) causes a change in the Sar I protein structure
and extends it though the membrane, causing it to bend
3. Sar I- GTP recruits other proteins that also increase the bending of the membrane
(protein-protein interactions)
➢ These other proteins (Sec23/24) also bind the cytosolic ends of the ER
transmembrane receptors to recruit cargo into the forming COP II vesicles
4. More
cytosolic COP II
proteins join the
complex (Sec
13/31)
➢ Forming the
outer structural
layer of the coat

Disassembly of COP II Coats

- Once the COP II coat is assembled,
the coated bud pinches off as a coated
vesicle
- GTP-hydrolysis triggers the release
of Sar I form the vesicle and therefore
all the other COP II proteins as well
b/c a loss of protein-protein interactions

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➢ Only GTP-bound Sar I is able to interact once it does its job, it interacts GAP,
loses Pi and fuses with Golgi
- Vesicle then fuses w/ Golgi
Targeted Vesicular Transport
*How do COP II vesicle know to fuse w/ Golgi?*
- Selective fusion maintains the directed flow of proteins through the cell
- Vesicles contain specific proteins that control its movement and fusion potential
- Two types of proteins are involved in targeting vesicles to specific compartments
➢ Rab proteins direct proteins
➢ SNAREs aid in the docking and fusion of vesicles
Tethering of vesicles
- Vesi les e o e tethered to
target compartment by extended Form link b/c
fibrous proteins and have same
multiprotein tethering type of
interactions
complexes
- Rab proteins are involved: small
GTP-binding proteins
➢ Associated to membranes by
a lipid anchor (look for
binding patterns)
➢ Over 60 diff. Rabs
➢ Specific Rabs associate w/
specific
membranes/organelles
➢ Recruit tethering proteins
from the cytosol
- Summary: Rab proteins are GTP binding proteins that are anchored to membranes using
lipid anchors direct vesicle to destination and are tethered to target using fibrous
proteins and multiprotein tethering complexes SNAREs help dock and fuse vesicle to
target membrane

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Docking of Vesicles
- The process that leads to vesicle fusion
- Requires interaction between membrane proteins on the
vesicle and on the target (recipient) membrane
- These membrane proteins are members of the SNARE
protein family
➢ vSNAREs on vesicle membranes
➢ tSNAREs on
target membranes
- specific tSNAREs
are on specific
membranes (similar
to Rabs)
- interaction
between v and
tSNARES pulls the
respective
membranes very
close together
- interaction between the SNARE proteins
allow for the extensive bending of the
membrane that then allows for the
fusion event
- Botulinum toxin snaps V&T snares,
does ’t allow for uscle co tractio
The Endo Membrane System
ER ERGIC GOLGI PM
- ERGIC = ER-Golgi intermediate
compartment
- The Golgi is located adjacent of the
nucleus and has a ribbon like
appearance (many stacks)
- Consists of many stacks of membranous cisternae membranes surround a lumen
- Golgi has several functionally distinct compartments (non-uniform from one end to
other)
- There is a progression of proteins from cis medial trans golgi network
➢ And there are distinct proteins residing in each compartment
Golgi function
- Primarily is a processing plants
- Proteins enter from ER to Golgi move from cis to trans face (have protein sorting
done in the CGN and TGN)
- Proteins are processed (eg. Cleaved like insulin) and modified (eg. Glycans) and sorted in
the different stacks of the golgi
➢ Some are sorted away to be sent to diff. vesicles that will go to diff. organelles

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Cis-Golgi Network (CGN)

- A network of tubules
- Newly synthesized secretory pathway proteins arrive at the Cis-golgi from the ER in
this stack, the proteins are sorted to determine which ones need to be brought back to
the ER vs ones that will move on through the Golgi
➢ Use signals
Trans-Golgi Network (TGN)
- A network of tubules and vesicles
- Sort proteins that are headed to the plasma membrane or to various intracellular
organelles
➢ Use signals
Protein Movement Through golgi
- 2 models:
• Vesicular transport Model
➢ Stacks are stable structures and the vesicles bud from one
compartment and fuse with the next
➢ Move non-resident proteins forward anterograde movement
➢ This changes the composition of each of the cisternae
• Cisternal Maturation Model
➢ Cisternae formed by transport vesicles that originate in the ER
➢ Cisternae physically move from the cis-end to the trans end
✓ i.e are not stable structures, and that they physically move from
end to end
➢ change their composition as they move mature
➢ Resident proteins are retrieved back from the stacks retrograde movements
- Find that when transport vesicles from ER to Golgi are blocked, Golgi disappears!
N-linked Glycosylation in the Golgi
- N-linked glycoproteins: sugars are trimmed and other sugars are added referred to as
complex N-linked glycosylation
➢ Required for proteins to achieve their final functional state
➢ Done by glycosyltransferases present in sequential stacks (assembly line)
- Trim mannose add N-acetylglucosamine trim mannose add N-
acetylglucosamine add fucose and galactose add sialic acid

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Glycosylation in the Golgi: O-linked

- O-linked glycosylation: sugars are attached to serine or threonine on specific proteins
➢ Happens specifically in the Golgi
➢ Proteins are already folded before this modification is made
✓ Possibly the proteins are recognized based on their shape
The Secretory Pathway secretion is the default pathway
- Goes form ER Golgi secretion (if no other signals present)
Types of Secretion
- Two Types of secretory activity: Constitutive and regulated
- Constitutive secretion: synthesis and secretion of proteins continuously, in an
unregulated manner
➢ Most membrane proteins and proteins that make up the ECM
- Regulated secretion: proteins to be secreted are densely packaged into large vesicles
called secretory granules that wait for a cellular signal before they fuse with the PM and
release their contents
➢ Cellular signal could be a stimulus (eg. like calcium ion concentration for neuro.T)
• Eg. Hormones, neurotransmitters, digestive enzymes
Exocytosis: final stage of secretion
- The process by which the contents of secretory vesicles are discharged
into the ECM
- The luminal part of the vesicles now become part of the extracellular
space
➢ Soluble proteins are
released
➢ Membrane proteins
become insert into the PM
➢ Sugars on the membrane proteins
now face outside the cell
How do Proteins Maintain Orientation during
Secretion?
- Transmembrane proteins are
synthesized in the ER
- Maintain orientation throughout the
endomembrane system
- Luminal side is accessible for
glycosylation and other modifications
➢ Could also be lipid-linked proteins
(eg. PrPc)

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Protein Sorting
- Many proteins are resident in the ER, the golgi, or in
lysosomes/mitochondria/chloroplasts/vacuoles
- Protein sorting in the cell: uses signals on proteins recognized by sorting factors
similar to the recognition/sorting done by the SRP
➢ Secretion is the default path ay … a y here else = requires signals
Protein Sorting: ER Resident Proteins
- As tra sport fro the ER to the Golgi is part of the default path ay follo ed y
proteins, ER Resident proteins contain a retrieval signal
➢ Retrieval signal allows for detection of proteins containing this signal in the Golgi
and their retrieval back to the ER
- Signal is present on both soluble and membrane proteins
➢ Retrieval signal on soluble proteins = KDEL (lys-asp-glu-leu) detected by the
KDEL receptor
➢ Retrieval signal on membrane proteins = KKXX (lys-lys-any-any) retrieved by
binding directly to the COP I coat
✓ Binds to the cytosolic side of COP I (Golgi ER) and comes back to ER
Protein sorting: soluble ER resident proteins
- ER resident proteins bearing the KDEL sequence move
from ER Golgi in the COP II vesicle
- KDEL receptor contains a KKXX motif and can bind directly
to the COP I coated vesicles
- KDEL-containing soluble proteins bind the KDEL receptor
and are thus moved back to the ER
- Summary: KDEL seq. containing proteins accidentally
move to Golgi interact with KDEL receptor (that has a
KKXX motif) that allows it to bind directly the COP I coat
after the KDEL protein binds with the KDEL receptors, the
KDEL receptors directly bind with the COP I coat (bind to the
cytosolic side) and return back to the ER
Protein Sorting: COP I Coat
- COP I coat is similar to the COP II coat
- GTP binding proteins are involved
➢ Moves proteins from the Trans to the Cis end of
the Golgi
➢ Moves proteins from Golgi to the ER

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Lysosomes
- Protein traffic form the TGN constitutive/regulated secretion, and
lysosomes/vacuoles
- Lysosomes are digestive organelles of animal cells, range in size and are bags of
hydrolytic enzymes (>50 enzymes)
- Enzymes = acid hydrolases work opt. at acidic pH (4.6 pH) and can break down any
type of macromolecule
- Have lots of proton pumps (pump H+ into lumen and increase the pH)
- Lysosomes function to: break down Extracellular material brought into the cell by
endo/phagocytosis = phagolysosomes
➢ Also break down organelles – autophagy autophagolysosomes (old organelles
are broken down - lysosomes are crucial in
Double stranded
memb. From ER
degradation pathways)
to create Lysosomes and Autophagy
autophagosome
- Will fuse with lysosomes
- Digest the organelles
- Cells do this more if they are starved of
nutrients provides energy
Lysosomes Digestion
- Macromolecules/organelles exposed to acid
hydrolyses get digested into small by-products
by products are transported to cytosol and
used by the cell
- - undigested/unwanted break down
products can:
1. Accumulate in the lysosomes (unsoluble
stuff)
2. Be removed through exocytosis (if build up
is too much)
- specialized lysosomes functions
➢ can fuses with PM in certain cell types
digest macromolecules that make up the
ECM in the cell exterior
✓ eg. Osteolclasts lysosomes release
enzymes that degrade bone // sperm
cell specialized lysosomes called
acrosomes fuses w/ PM and releases
e zy es that degrade the egg

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Protein Sorting: Lysosomal Enzymes

- Ho do a id hydrolases get i to the lysoso e?
- synthesized in the ER glycosylated in the ER (N-linked glycan) transported to Golgi
specific mannose sugars of the N-linked glycans on acid hydrolases are
phosphorylated by enzymes in the cis-golgi
➢ create: Mannose-6-phosphate
- Mannose phosphorylation = signal for transport to lysosomes (on N-glycans)
- Summary: proteins destine to go to lysosomes are made in the ER, have a N-linked
sugar added in the ER as well go to Golgi and the mannose sugars on the N-linked
sugar are phosphorylated to create Mannose-6-phosphate which is the signal to get
transported to the lysosome
- The mannose phosphorylated lysosomal proteins are recognized in the Golgi by
Mannose-6-phosphate receptors MPRs
➢ MPRs are transmembrane proteins that reside in the TGN
✓ Bind proteins containing the Mannose-6-phosphate signal
✓ MPRs bound to proteins w/ the
Mannose-6-phosphate signal then
bud into Clathrin coated vesicles
✓ Those vesicles fuse with endosomes
✓ Endosomes mature into lysosomes
containing acid hydrolases

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Clathrin Coated Vesicles

- Clathrin: protein structure
made of 6 proteins (subunits)
➢ 3 light chains and 3 heavy
chains and assembles into
a triskelion structure
➢ comes together to form a
basket type structure
- Outer shell: structural
assembly of Clathrin form a
honeycomb lattice
- Inner Shell:
➢ “e eral adaptor protei s i d to Clathri o o e e d a d to argo
re eptors o the other side
➢ Diff. adaptors for diff. organelles (eg. GGA for transport from TGN to
lysosomes)
G- ➢ diagram shows GGA adaptor linking to transmembrane protein and to
protein the clathrin coat
➢ binding GTP is first step to assemble Clathrin coat binding of GTP
activates vesicle

Something Else to Think About

- I-Cell Disease
➢ Lysosomal storage disease where mannose-6-phosphate pathway does ’t work
➢ Results in severe skeletal and developmental abnormalities
➢ Mutation in gene that codes for a protein involved in the mannose phosphorylation
of proteins = no M6P = no signal = no trans port of acid hydrolases to lysosomes
✓ Lysosomal enzymes are transported to the cell exterior constitutive
secretion
✓ Material to be degraded accumulates w/I swollen lysosomes swell w/
undigested materials
✓ Essentially a defective gene in the cis-golgi

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Vacuolar protein sorting

- Vacuoles make up 90% of volume in plant cell and are similar to lysosomes (do the
digestive work)
- Vacuolar proteins have a signal sequence (RER) proteins targeted to RER are
translocated into ER and signal is cleaved
➢ Have a secondary signal that moves them through the secretory pathway to the TGN
in the TGN the vacuolar targeting sequence (VTS) is recognized (can be at either
terminal)
➢ Results in the vacuolar proteins budding off from the TGN and being targeted to the
vesicles vesicles fuses w/ vacuole and deliver proteins to this compartment
➢ VTS is then cleaved
- Essentially: 2 signal sequences involved signal sequence (to go to RER) and VTS
Protein Sorting to Other Organelles
- Protein sorting to peroxisomes/mitochondria/chlorosplasts also involve protein signals
that are recognized by receptors that then transport them to their correct
compartments
- Protein transport (import) to these organelles occurs post-translationally (after they are
synthesized on free ribosomes on the cytosol)
➢ Proteins are synthesized on free ribosomes on the cytosol
• Different from ER, which imports proteins co-transionally
Protein sorting to Peroxisomes
- Peroxisomes: simple membrane bound organelles that break down H2O2 in an oxidation
reaction using the enzyme catalase
➢ Site of synthesis and destruction of hydrogen peroxide (H2O2)
➢ Also break down fatty acids
- Peroxisomal proteins have signals that direct their transport to peroxisomes: import
proteins to their native state
- Peroxisomal transport signal (PTS): on soluble (luminal) Peroxisomal matrix proteins
- Membrane Peroxisomal
transport signal (mPTS): on
Peroxisomal membrane proteins

- Summary: Proteins containing

transport signal are synthesized
on free ribosomes on cytosol
receptor binds signal
accompanies cargo to
peroxisomes and the receptor
temp/ interacts w/ import translocon on Peroxisomal membrane receptor becomes
part of translocon which then allow entry of cargo protein into peroxisome
➢ Peroxisome receptor then returns to cytosol
➢ Proteins are imported in their fully folded form

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LECTURE #5 ENDOCYTOSIS AND PHAGOCYTOSIS

Endocytosis
- The process by which cells internalize ECM proteins, cell
surface receptors and proteins bound to the PM
- Two Types:
➢ Bulk phase pinocytosis (cell-drinking
✓ All materials present in the ECF is taken up
into the cell in a non-specific manner
✓ i.e internalization of dyes cell samples its
surroundings to get info of its surrounds
➢ Receptor mediated
✓ Provide a selective way to internalize EC molecules by choosing to bring in
receptors that bind those molecules (and are choosed based on cell surface
molecules)
✓ Can take up hormones/growth factors or other signaling molecules by their
binding to cell surface receptors that are then brought in by the RME
✓ Receptors becomes concentrated in the vesicles (10-20x higher
concentration than on the rest of the membrane)
Endocytosis: Receptor Mediated
1. Cell surface receptors bind the
extracellular ligands (i.e growth factor
binds to receptors and sends signal to
cell to undergo cell division // when not
needed, entire receptor is swallowed)
2. Clathrin binds the other end of the
receptor (cytosolic side)
3. Clathrin-coated pits form and bud
inwards form the PM
4. Pits containing receptor-ligand complexes
invaginate into the cytosol and pinch free of
membrane become Clathrin coated vesicles
➢ GGA adaptors are used for Clathrin coated
vesicles from TGN to lysosomes
➢ AP2 adaptors are used for Clathrin coated
vesicles that bud from plasma membrane
✓ AP2 is a multi-subunit adaptor that binds
the transmembrane receptor w/ the
Clathrin coat
✓ Isn’t al ays acti e activated when
interacts w/ phospholipids

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➢ Dynamin is required for the fission of the Clathrin coated vesicle from the
membrane
✓ Dynamin = GTP-binding protein
✓ Forms Helical collar around the neck of the coated
pit
✓ GTP hydrolysis coated vesicle is severed from
the membrane

5. Clathrin un-coats from vesicle within

minutes of internalization (GTP hydrolyzed,
Clathrin pinches off)
6. Vesicle forms an early endosome (EE, fuses
in SC)
7. EE Matures into sorting compartment (SC);
lumen acidifies by action of proton pumps
dissociate ligand and receptor
8. Receptors recycle back to surface vesicles
9. Endosomal carrier vesicles (ECVs) leave the
SC and fuses w/ late endosome (LE)
10. LE mature into lysosomes (maturation step)
11. Endocytosed material is broken down by
acid hydrolases in the lysosomes

Phagocytosis
- Cell eati g
- done by many single-celled protists
- is a protective mechanism rather than a
way of feeding in mammals
- it is a form of endocytosis carried out by specialized cells eg. Macrophages
- receptors on immune cells bind to dead cells, bacteria etc.

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Phagocytosis: macrophage
- not all bacteria are killed by the
process of phagocytosis in macrophages
- some bacteria can secrete proteins
that interfere w/ the fusion of the
phagosome w/ lysosomes
(phagolysosome)
➢ allows bacteria to continue to live
and multiply in the phagosomes w/I the
macrophages (eg. Mycobacteria,
salmonella)

The Plasma Membrane: Structure and Function

- is about 5-10nm
Functions of the Plasma Membrane
1. Compartmentalization: encloses the contents of the cell
2. Serves as a scaffold for biochemical activities (eg. Signaling proteins)
3. Is a selectively permeable barrier (prevent unrestricted exchange of molecules w/ the
extracellular space)
4. Transports solutes (moves ions/AA/sugars etc.)
5. Allows the cell to respond to extracellular signals
➢ Cell surface receptors bind ligand on cell exterior can send signals into the cytoplasm
of the cell i.e signals could tell cells to undergo CD growth factor)
➢ ECM proteins also interact w/ cell-surface receptors
6. Intercellular interactions allows exchange of materials and info. w/ neighboring cells
➢ Cells can adhere or form junctions as part of this interaction (eg. Synapse)
History
- Ernst Overton fou d that aterials e teri g the ell first dissol e i the outer
ou dary layer
- Placed plant root hairs in diff. solutes only lipid soluble solutes could get into the cell
led to conclusion that membrane has fatty oil components
- Gorter and Grendel
➢ Lysed RBCs, spread their lipids over measured 2x as much lipid as the predicted
surface = PM is a bilayer
- Polar groups of the lipids would interact w/ water; hydrophobic groups would interact
w/ eachother (hydrophobic interactions)
- Davison and Danielli: proposed proteins are in the bilayer

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- Singler and Nicolson: Fluid-Mosaic Model

➢ Fluid: indiv. Lipids can move laterally within the bilayer
➢ Mosaic: proteins are located w/I the lipid layers PM is a dynamic structure
Plasma Membrane Structures
- 2 layers contain diff. types of lipids = asymmetric distribution
- ratio of protein:lipid in the PM is dependent on the cell
➢ i.e nerve cells have lots of lipids, very few proteins
- PE lipid allows for protein bending
Membrane Lipids
- Membrane lipids are amphipathic and have 3 main types
1. Phospholipids
2. Sphingolipids
3. Cholesterol
Phospholipids
- Composed of a phosphate group, glycerol, 2 fatty acids
- A polar group linked to negatively charged phosphate
group (can be choline/serine/inositol/ethanolamine)
➢ Small hydrophobic groups
➢ High water soluble ends
Sphingolipids
- Composed of: an amino alcohol with a long, hydrocarbon
chain = sphingosine
➢ Have a single fatty acid chain (that give it its identity)
- The fatty acid chain is attached to the amino group of
sphingosine (this is a ceramide)

- additional groups can be added

onto the terminal alcohol of
sphingosine
➢ + phosphoryl choline =
sphingomyelin
➢ + sugars = glycolipid (eg.
Cerebroside, ganglioside)
- glycolipids are prevalent in the
nervous system myelin

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Cholesterol
- cholesterol is a sterol molecule
less amphipathic (mostly hydrophobic
rings)
- very small hydrophilic (hydroxyl)
group faces the membrane surface
- the hydrophobic rings are flat & rigid
interfere with the movements of the
fatty acid tails
- rigidity of cholesterol prevents movement
Membrane Proteins
- 3 Types
1. Integral Proteins transmembrane proteins
2. Peripheral Proteins cytoplasmic or extracellular side of PM (associated w/ PM but
are not embedded)
3. Lipid-anchored proteins can be on cytoplasmic or extracellular side
1. Integral Proteins
- Transmembrane proteins
- Can pass through the membrane once or several
times
➢ Single or multi passage
- Amphipathic and can form channel for passage of
solutes
➢ Some hydrophilic resides placed amongst
hydrophobic region (to allow soluble things to
come in and out through its channel)
- Have the ability to move laterally through the membrane (not fixed, extracted by
detergents)
2. Peripheral membrane proteins
- More loosely associated w/ PM than integral
membrane proteins have electrostatic interactions
- Can associate w/ the lipids (hydrophilic regions) OR
with the integral membrane proteins in the PM
- Often transiently interact with PM eg. Respond
to signals
- On the cytosolic face = provide structural support
to PM
- Solubilized from PM by high salt solution

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3. Lipid Anchored Proteins

- Associate dw/ PM by means of a lipid group
- On the extracellular side, proteins are attached by a
complex oligosaccharide linked to phosphatidylinositol
- Glycophosphatidylinositol linked proteins (GPI-anchored
proteins)
➢ Eg. Some receptors, prion protein
- On the cytosolic side, proteins are attached by long
hydrocarbon chains embedded in the inner leaflet of the PM
➢ Eg. Certain enzymes, signaling proteins
- Whole protein enter the RER, once fully synthesized and
has sequence on C-terminus protein adds the anchor and
its moved to the PM
➢ sugars are added on the C-terminus

Membrane Carbohydrates
- short, branched hydrophilic oligosaccharides (~15 sugars)
- glycolipids/proteins
- face extracellular space and
are important for
communication
- aid in cell-cell interactions
- ca deter i e the cell’s
identity
- carbs attached to membrane
lipids on the surface of RBCs
determine our blood groups

- the lipid bilayer exists in a relatively-fluid

state flip flop (can have changes in the
sides of the bilayer)
➢ transverse diffusion
➢ lateral shift
- the 2 leaflets of the PM have distinct
lipid compositions
➢ top is phosphtidylcholine and
sphingolipids
➢ cytosolic side is diff.

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Why Fluid?
- Fluidity allows for: membrane fusion events, molecular interactions
➢ Eg. Endocytosis, cell-cell interactions, cell movement
- Membrane Fluidity depends on: temperature, types of lipids, presence of cholesterol
(disrupts packing of fatty acid chains)
- Unsaturated fatty acids pack together less well than saturated ones (unsaturated=
double bonds)
➢ The greater the degree of unsaturation, the
lower the temp. before the bilayer gels =
transition temperature

Integral Proteins fluidity

- integral proteins also move in
the membrane (A)
- various factors influence their
mobility:
1. Local Crowding of Proteins
(D)
➢ Neighbors restrict
movement
2. Barriers on the cytoplasmic
side of the membrane (B, C,
&, E)
➢ Interactions with the
membrane skeleton on the
cytosolic side
➢ Interactions with peripheral
membrane proteins
3. Interactions with EC material (F)
➢ Entangled w/ ECM components

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Lipid Rafts
- Membrane micro domains (small regions of the PM)
enriched w/ sphingolipids, cholesterol, and GPI-
anchored proteins
- These domains are more highly ordered than their
surrounds fluid membrane rafts
- Can concentrate specific proteins (eg. Signaling
molecules)
- Functional compartments of PM
- Very little endocytosis

Transport across the PM

- Dual function of the PM:
➢ Keep
contents in the
cell
➢ Allow exchange of materials into/out of
cell
- Movement of Substances across the PM
occurs by:
1. Simple Diffusion across the lipid bilayer
if lipid soluble
2. Simple diffusion through protein channels
3. Facilitated Diffusion by a protein
transporter larger molecules
4. Active transport energy coupled process

The Movement of Solutes

- Diffusion: substances moves from region of high to low concentration
➢ Spontaneous, follows chemical gradient
- If substance is charged (ion), diffusion also affected by the difference in charge between
the compartments
➢ Opposites attracts
➢ Energetically favored if compartments have opposing charges
➢ Electrical potential gradient
- If both factors involved electrochemical gradient
- Factors that influence simple diffusion across the PM
1. Polarity of the solute more lipid soluble = faster diffuses across the PM
2. Size of the solute small, uncharged molecules penetrate faster, large have poor
per ea ility eg. “ugars, AA do ’t go through
- Water partition coefficient measures lipid solubility

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Water?
- Water readily diffuses through the PM
(quicker than ions/polar solutes)
- Water moving from low [solute] to high
[solute] = osmosis

Simple diffusion through Protein channels

- Ions move through protein channels (ion
channels) in the PM
➢ Bi-directional movements
➢ Channels are ion specific (and move down
gradient)
➢ Are gated ; either in an open/closed state
- Types: voltage gated (opens on charge diff.) // ligand-gated (opens upon binding of
ligand)
Check ion identity
Voltage-gated ion channels
- Open depending on the difference in ionic charge
between the 2 sides of the membrane (eg. K+ ion
channel)
➢ Complex multi subunit complexes
➢ Highly selective ion filter
➢ Proteins change conformation to allow entry of
ions into the cells require a charge

Gate opens from interaction

btwn filter and helices

Ligand-gated ion channels

- The binding of a specific molecule opens and closes the
channel to ions
➢ Ligand itself does not through the channel
➢ Ligand can bind to either the inside or the outside
parts of the channel cytosolic/extracellular
surfaces
• Eg. Acetylcholine neuro.T binds to the
outside of the channel
• Binding of acetylcholine to the binding site
causes a conformational change that open the
channel and allows in

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Facilitated diffusion through the PM

- The diffusing substance uses a transmembrane protein to facilitate its diffusion across
the bilayer facilitative transporter
- Binding to the transporter on one side of the PM causes a conformational change in
the protein such that the bound substance now gains access to the other side of the PM
- Transporters
➢ Operate passively transport substances equally well both directions
➢ Relative [ ] of substances on either side of PM determines the next direction of
movement
➢ Are specific for molecules they transport
• Mediate entry of polar solutes like sugars and amino acids
➢ Do not require energy
➢ Activity can be regulated
➢ Transport molecules
Eg. Glucose Transporter
➢ Facilitates diffusion of glucose from
the bloodstream into the cells
➢ Glucose binds to one of several
glucose transporters dep. On the type of cell
(GLUT 1, 2 in humans)
➢ Glucose is phosphorylated in the
cytosol keeps the intracellular glucose
concentration low allowing for diffusion
into the cell
• Phosphorylation changes glucose so
that the he i al gradie t of lo o . Of
glucose in cell remains

Regulation of glucose transporters

- Blood sugar level low Insulin low GLUT 4
in cytoplasmic vesicles is endocytosed
➢ Few transporters on PM, very little
internalization of glucose into cells
- Increase in blood sugar level (eg. Eat a donut)
insulin high (secreted) insulin binds to insulin
receptors on PM sending signals for vesicles to
exocytose GLUT 4
➢ Exocytosed GLUT 4 = more transporters on PM = more internalization of glucose into
cells

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- Diabetes = defective insulin receptors or defective GLUT 4 transporters

Active Transport
- The movement of ions against the concentration gradient ions moved in only 1
direction in a gradient
- Active transport is carried out by proteins that transport the ions pumps
➢ They are integral membrane proteins that bind specific molecules only
➢ Binding of the molecule to be transported to the pump results in a conformational
change that allows molecules to be delivered to the other side of the membrane
• Similar to facilitated transporter
- Process requires energy ATP
➢ Phosphorylating and dephosphorylating causes changes in shape
- Eg. Proton pumps on the lysosomal membrane
Eg. Sodium-Potassium pump (Na+/K+-ATPase)
- Responsible for: excess Na+ ions outside
the cell, excess Ka+ ions inside the cell
- Pumps Na+:K+ in the ratio of 3:2
➢ i.e pumps out 3 Na+ and brings in 2 K+
➢ found only in animal cells
Process:
1. 3 Na+ ions are binded to the cytosolic side
of the PM
2. Pump is phosphorylated by ATP hydrolysis
3. Conformational change causes
binding site to face cell exterior and Na+
ions are released to ECF
4. 2 K+ ions bind on the extracellular
side
5. Pump loses phosphate (loss of
phosphorylation), pump is restored
back to original shape
6. Pump is restored back to original
shape (after losing phosphate) K+
ions are released into cell interior

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Something else to think about

- A neuro.T called glutamate
can activate AMPA and NMDA
receptors in the brain involved
in memory formation through
long-term potentiation (causes
changes in synaptic linkages)
- weak memorization only
allows a few AMPA receptors to
be activated by glutamate
(Allowing Na+ in neuron), NMDA is
inhibited by Mg2+
- good memorization allows
AMPA AND NMDA receptors to
both be open, and allows influx of
sodium AND calcium calcium
acts as 2nd messenger triggering
long-term memory recollection

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LECTURE #6

STUDYING PROTEINS

- how do we look at proteins?

Fluorescence Microscopy
1. Immunofluorescence
2. GFP technology

Fluorescence Microscopy
- Use a fluorescence microcope and detect
the location of compounds known as
fluorophores
➢ Fluorophores/dyes absorb energy of a
specific wavelength (UV) and release a portion
of this energy as longer, visible wavelengths of
light known as fluorescence
• e- goes to higher energy level, when it falls
back, it release light and the machine detects
the fluorescence signal
Fluorescence Microscope
- light source
- Filters (I) allow only light of the wavelength that
excites the fluorophore to reach the specimen
- Specimen fluoresces
- Filters (II) allow only the fluorescencent light
(LONGER wavelength) to reach the eyepiece
- Object appears brightly coloured against a dark
background

Fluorescencent dyes
- A fluorophore is attached to an antibody that is
directed against a specific cellular protein
immunofluorescence

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Antigen bind to specific B-

cell antibody B-cell
divides a lot and secretes
lots of an

Immunofluorescence: Antibodies
- Antibodies: IgG
• Has 2 light chains and 2 heavy chains that are connect by hinges
➢ Bind very specifically to proteins antigens
➢ Made by vertebrae as a defense against infection
➢ Made by B cells in the blood (a type of WBC)
➢ Each B cell carries a unique type of antibody on its surface that reacts against a
specific antigen
➢ When an antibody encounters its antigen, the B cell divides rapidly and secretes
lots of antibodies into the bloodstream

How to make antibodies for the Lab?

- Inject human proteins (eg. Protein human actin) into lab animal (eg. Rabbit)

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➢ Purify and inject antigen into animal B cells make antibodies against the antigen
lots of Ab present
- Che k a i al’s lood for evidence of Ab production
against the antigen
- Boost the a i al’s i u e syste se eral ti es
w/ antigen injection over weeks
- When a large # of antibodiesa re made by the
animal (high titre), harvest the animal and use its blood
as a source of Ab
➢ Rabbit anti-human actin Ab OR rabbit anti-actin
Ab
➢ (animal injected _ anti-[type of animal antigen
POLYCLONAL
taken from] _ name of antigen _ Ab)
ANTI-SERUM
NOTE: many diff. B cell Abs may fit different parts of the
same antigen these are called epitopes
➢ epitopes = a protein a has many antigenic
components and B cells may produce Abs against each
of these components
the blood obtained by these mice is a pool of many
diff. Abs that all react against the injected protein,
creating a Polyclonal antiserum

- we can make a Monoclonal

serum that are derived from a s
ingle clone of B cells that
produce Abs against one of the
antigenic components
➢ harvest spleen, find the B
cell that creates the right
antibody and fuse w/
tumour cell to create a
hybridoma create
infinite amount of Ab
- adv. Of monoclonal Abs is that
there is an unlimited supply of
them from the hybridomas
➢ eg. Can screen PrPc and
PrPsc by detecting
monoclonal Abs specific to
one of them
➢ PrPc = ℎ���� �� , � �sc = ℎ�� Abs

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- spe ifi ells a ’t gro i HAT edia

- harvest cells form spleen, and fuse w/ myeloma cells, then
grow the cells on selection medium (HAT)
- myeloma cells (black) die on HAT medium, spleen cells
(red) die outside of natural env.
➢ Are left with hybridomas these hybridomas are then
screened to find which one produces the desired antibody
- Can now covalently link fluorophore to the antibody
specific for the protein of interest cells are fixed and stained
w/ this antibody
then view cells under a fluorescence microscope
fluorescencent antibody will localize the protein w/I the cell =
immunofluorescence

Direct vs Indirect
Immunofluorescence
- Direct = antigen binds to
antibody
- Indirect = instead of direct interaction w/ antigen and antibody, have 1* antibody and
* a ti ody, su h that the * a ti odies’ a tige , is the * a ti ody
➢ Indirect is cheaper, and makes a more visible fluorescence

GFP Technology
- The GFP: green fluorescencent protein is form the jellyfish
- Recombinant DNA is made by attaching (fusing) the gene that codes for GFP w/ the
gene of the protein of interest fuses the gene coding for a certain protein
- This DNA is introduced into cells in culture transfection
- DNA RNA protein
- Cell ill sy thesize a hi eri protei ith GFP fused to protei of i terest i.e actin
fused w/ GFP)
➢ Can now visualize the protein by using the wavelength of light that excites GFP
• Known as GFP-fusion proteins

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GFP-Fusion Proteins
- Ca look at li e ells and follow protein dynamics
➢ For immunofluorescence, cell has to be dead and permeable for the Ab to enter it,
GFP tech. allows us to see the cell alive
- Various variants of GFP have now been
engineered and glow in different color
picture shows cis-ternal maturation model
of the golgi
➢ Shows the cig-golgi (green) becoming trans-
golgi (red) in the 3rd and 4th frames

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Confocal laser-scanning microscopy

- Specialized type of fluorescence
microscope also used
flurophores
- Uses a laser beam (instead of light)
to create an image instead of a
UV lamp
- Laser is used in combination w/ a
pinhole that allows us to optically
section through the specimen
focus on specific focal planes
- Avoids the blurry images that we
sometimes get from normal
fluorescencent scopres
➢ Light emanating from above
and below the plane of focus do NOT form the image
• Reason image is blurry is typically blurry for fluorescence microscopes is b/c
the ’re taki g a average of diff. pla es, thus creati g glare

STEPS
Adj. pinhole allows light from only one
1. Light from laser
focal plane to come in
passes through
aperture
2. Light is reflected
towards objective lens
using dichroic mirror
3. Laser excites diff.
fluorophores from diff.
focal planes, and their
light is reflect through
the dichroic mirror
Mirror preve ts laser’s light fro 4. Pinhole only lets in
being reflected back up, and only
fluorescent light form diff. focal light from one focal
planes comes up plane (plane of focus) at
a ti e, so do ’t get light
from diff. parts of
specimen
5. PMT enhances and
collects photons
6. Computer generates image

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FRAP
- Fluorescencent Recovery After
Photo bleaching
- Steps
1. Label proteins w/ fluorophore
2. Bleach a specific area with a laser
and remove the fluorescence from the
proteins in that area
3. Recovery look to see if the light
ree ters that photo lea hed area
➢ If light reenters area quickly, then
the protein of interest is very mobile

Photoactivatable GFP and super

resolution microscopy

- Photoactivatable GFP allows us to

manipulate when we want to activate
GFP to have fluorescence
- STORM: Stochastic optical
Reconstruction Microscopy
➢ only activate GFP in location we
want to observe
allows to activate specific regions of
the molecule we want to image, and
prevent getting a fuzzy picture from
proteins that may be close by
➢ end up photo-activating different
regions each time, and then
reconstructing image that fits all the
regions together

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Protein Purification I

- Process involves:
1. Cell Fractionation
• Breaking open cell contents (cell extract)
• Separating the contents of the cell (cell fractionation)
• Break open cell and separate crude contents
2. Protein Separation
• Using a column containing a solid matrix (Chromatography)
OR
• By applying an electric field to a solution of
proteins (Electrophoresis)
Cell Fractionation
- To get at specific proteins that may be
located within specific cellular organelles, we
must first break open cells to release their
contents
➢ Referred to as making a cell e tract or
homogenate
- Release everything from the inside of the cell collect compounds or proteins
- What type of extract method determines what is the end product
- Separate extract into its components: organelles or groups of proteins
➢ Fractionate = the extract into its components
✓ Cell fractionation
- Process: take cell break open
make mixture crudely separate
the extract into its crude components
separate by different properties
Making a Cell Extract
- Break open cells in a controlled
manner
- Use techniques that break open the
plasma membrane and release the
contents of the cell
- Many possible methods
➢ Example:
1. Use mild detergent to make
holes in the membrane detergent disrupts all phospholipid bilayers
(depending on its concentration and type)
2. Shearing membrane by force using a tight fitting plunger and glass tube
• Crush the cells against glass and break them, to get the compounds must be
carried out in a controlled manner to account for all proteins
➢ Eventually end up with cell extract w/ cytosol and organelles

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Centrifugation
- Proteins of different size and density in a solution will settle at different rates in a tube
b/c gravity
- Centrifugation: forces that are several times that of gravity are applied to solutions to
increase the rate at which the different components will settle into the tube, based on
size and density
➢ Done in a centrifuge
• Eg. Nucleus is different from
the mitochondria will have a
different size and density after
centrifugation
- Microfuge: 1-2 mL samples,
15k gs of force, nuclei
separation
- Ultrafuge: 15-25mL samples,
150k gs of force, organelle
separation
- larger, denser components
sink fastest to the bottom
pellets
- smaller, less dense
components stay in suspension
above the denser material
supernatant
• process is repeated
multiple times
- the cell extract can be
centrifuged at
progressively higher
speeds to separate the
cellular components
based on their size and
density
➢ called differential
centrifugation

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Density Equilibrium Centrifugation

- cellular components can be separated based on their buoyant density
➢ put through a steep density gradient of sucrose
➢ centrifugation will make the components move to a point in the sucrose that
matches their density they will stay there
➢ different components will form bands that we can collect into separate tubes
➢ Highest density = bottom of tube, lowest density = top
Summary: having a
gradient of sucrose
apply extract to the
sucrose gradient
centrifuge after
centrifugation, cellular
components will move
to the part of the
gradient that matches
their density will
create distinct bands
How do we get the separated components?
- Cell Fractionation cellular
components are separated
into fractions (individual
amount of volume)
- Summary: poke a hole in
bottom of the tube allow
for certain mL of what want
allow contents of the
centrifuge tube to be dropped
in other racks (depending on
how much volume) test
the tubes for presence of our
desired cellular component

Protein Separation
- Use a column containing a solid matrix (chromatography) OR apply an electric field to
a solution of proteins (electrophoresis)
- Proteins different in many ways
➢ Size, shape, charge, hydrophobicity, affinity to bind certain molecules eg.
Ligand/receptor interactions
• Use these differences to separate proteins from eachother

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Protein Separation: Column Chromatography

- A column contains a specific type of
porous matrix a type of binding
substance (typically beads)
- pour the mixture of proteins over
the matrix in the column different
proteins in the mixture will interact
with or move through the column in
different ways (because of their
difference in properties)
- these difference in interactions will
slow down proteins to varying degrees
some will move faster than others
- collect fractions from the bottom of
the column into different tubes and
each fraction will have a different set
of proteins separated out of the
original mixture
- use a property that is distinct of red
vs green they interact different with
the matrix red elutes faster than green
Column Chromatography
1. Gel Filtration Chromatography separate based on size (using a gel w/ holes)
2. Ion-exchange chromatography separate based on charge
3. Affinity
chromatography
separate based on
ability to bind to a
proteins or an antibody
(immunoaffinity)
Gel Filtration Chromatography
- Separation of proteins
based on their size
we set the bead size
- Matrix is composed of
porous beads
proteins in the mixture
can move in and out of
the beads
➢ such that: large
proteins will move
around the beads
a d elute / a ’t e ter the eads
➢ small proteins will enter the beads and take a longer time to elute

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Ion Exchange Chromatography

- separate proteins based on ionic charge net charge of a protein is the sum of all the
charges of its amino acids
- matrix that is positively charged = anion exchanger // negatively charged = cation
exchanger
➢ proteins will interact or not interact w/ the matrix based ont ehir charged
• eg. If protein has lots of arginine, use certain types of +ve or –vely charged
matrixes protein will stay on or off dependings on the net charge from their
R-group
- Example: diethylaminoethyl (DEAE) cellulose positively charged binds negatively
charged proteins anion exchanger
➢ Need to apply a competition between the –ve and +ve charges
• Eg. NaCl solution will compete with molecules for charged sites and displace
them
• Proteins can be eluted using salt solutions or by altering the pH of the buffer
Summary: add mixture of different charged proteins to charged matrix proteins that have a
ertai harge ill e attra ted to the eads’ harge, protei s ot attra ted ill elute then
add a salt solution or change the pH of matrix to elute suspended proteins that are attracted
to the beads

Affinity Chromatography
- Separation of proteins based on interaction with
other proteins affinity
- Matrix is composed of beads with specific proteins
attached to it only protein of interest will bind to
the matrix associated proteins
➢ Eg. Acetylcholine receptor will bind specifically
acetylcholine on the matrix
- Essentially is placing a receptor that your protein
will bind to all other proteins will pass through and
elute
• Eg. Take matrix covalently attach insulin to
beads put into the column and insert mixture if
mixture contain insulin receptor, it will bind
- Use of salt salt is a polar molecule and can disrupt the protein-protein interactions
that allow the proteins to interact w/ each other bound ligand falls off its substrate

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Electrophoresis
- When proteins are in an electric field, they migrate at a speed based on their net
charged, shape, and size basis of electrophoresis
- Proteins in a solution are driven by an electrical current through a gel matrix matrix is
composed of cross-linked polymers of acrylamide (a sieve) called Polyacrylamide
gel electrophoresis = PAGE
- Matrix serves as a mesh that entangles proteins as they attempt to move through the
gel
How to Separate based on size (molar mass)
- Heat protein solution to denature all proteins mix with a negatively charged
detergent known as SDS (sodium dodecyl sulfate)
- SDS binds to all types of proteins
➢ Coats all with a layer of negative charge charge is not a factor anymore
➢ Number of SDS molecules bound to a molecule is proportional to its mass
➢ Bound SDS molecules create repulsion forces that change all proteins into a rod-
like shape shape is not a factor anymore
- SDS-PAGE = proteins separated solely based on their size
- Summary: denature proteins using heat add SDS detergent to proteins coats all
proteins with a layer of negative charges charges repel and create a rod shaped
molecule run through PAGE separate solely on size b/c # of SDS molecules
attached to molecule = molecular weight
Steps:
- Cell extract is used as starting material use heat
to denature and coat with SDS
- Polyacrylamide gel is polymerized between 2 glass
plates and placed in an apparatus between 2
compartments containing buffers
- 2 electrodes are placed on opposite ends
protein solution w/ SDS is loaded onto the top end
of the gel along with a visible tracking dye (usually
blue)
- voltage is applied and the proteins migrate
through the gel to the anode (+ve electrode)
➢ proteins separate by size
- gels can be stained w/ dyes to reveal the location
of the proteins
- smaller proteins move to the bottom end of the gel
(small molecular weight)
- larger proteins stay near the top part (large molecular weight)
- MIGRATE BY SIZE

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2-D Gel Electrophoresis

- 1-D only uses size
- a different method can be used to
separate proteins first by CHARGE (1st
dimension) and then by SIZE (2nd
dimension)
➢ very large numbers of proteins can
be separated using this technique
1000+ proteins in a mixture
• proteins are unlikely to have same
size AND charge
- a pH gradient allows proteins to
migrate until they lose all their charge
point where protein loses its charge is
called its isoelectric point
- Steps: synthesize ampholites (w/
diff. PI) and they orient themselves to
create a pH gradient proteins go their
optimal pH then so SDS page on the existing gel

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LECTURE 7: MITOCHONDRIA

- Mitochondria is not a simple circle, is in fact a network an aggregation network

Origin
- Arose from aerobic prokaryotes an Eukaryote ate an aerobic prokaryote
- Mitochondria can divide w/I the cell mitochondrial fission binary fission
- Mitochondria can also fuse within the cell mitochondrial fusion
➢ Balance between fusion and fission dictates mitochondrial numbers and length
➢ Typically see that fusion happens more often than fission
Mitochondrial Fission
- Mitochondrial fission induced by contact w/ thin tubules from
the ER that encircle the mitochondria
- ER tubules initiate constriction which is then completed through
the action of soluble, cytosolic proteins that are recuirted to the outer
surface of the mitochondrion
- Mitochondria has lots of affinity for the ER when conditions for
cell are not good, ER decides whether to have mitochondrial fission or
fusion
➢ Eg. When energy demand is high, might have fusion b/c larger mitoC = better
- Summary: where MitoC are split is determined by the ER tubules wrap around area
of mitoC fission (usually middle) brought by Rab proteins (recruited by Rab proteins)
they recruit more proteins from the cytosol form a ring snap through a similar
process to dynamin (for vesicles)
Mitochondrial Structure
- Outer Membrane (OMM) intermembrane space Inner
membrane (cristae folds) Matrix
- 2 membranes, 2 aqueous components (matrix and
intermembrane space)
- top of cristae are bumpy b/c have ribosomes and ATP synthase
molecules

OMM
- the outer mitochondrial membrane serves as the outer boundary
for the organelle and has a 50-50% protein to lipid ratio

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- has a large and diverse number of proteins that function as enzymes mitochondria
determines whether the cell dies (Apoptosis)
- has several large protein channels (Porins)
➢ Porins have: beta pleated sheets
➢ can allow for the passage of a large number of
molecules (eg. ATP)
➢ respond to conditions within the cell are very
regulated

IMM
- the inner mitochondrial membrane has
➢ cristae = thin folds
➢ has 75% protein-25% lipid ratio
• for selective transport function
• lipids are mostly cardiolipin (found on
bacterial membranes) and have no cholesterol
➢ have almost 100 diff. proteins
- is very impermeable to molecules and has many channels and pumps

Matrix
- has a gel-like consistency b/c high concentration of proteins
- contains own DNA mtDNA and ribosomes
➢ mtDNA encodes 37 genes
Where do the other proteins come from?
- Majority of proteins for mitochondrial function are encoded by nuclear DNA
- The proteins are translated on free ribosomes in the cytosol
➢ They are then imported into the mitochondria post-translational import

Signal for Import

- Matrix Proteins
➢ Signal = mitochondrial Pre-sequence
• Found at the N-terminus of proteins mostly positively charged residues
- IMM Proteins (integral membrane proteins)
➢ Contain internal targeting sequences and stop transfer sequences
• Stop transfer sequences are hydrophobic do ’t ha e ha ges, ut use otifs
➢ IMM proteins DO NOT have a pre-sequence
Import
- For mitochondrial import, proteins need to be in an unfolded state helped by
chaperone proteins in the cytosol like Hsp70 to stay unfolded
- Unfolded proteins then bind to membrane receptors on OMM (using signals)

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- Receptors are located close to large protein channels called TOM Complexes (eg. Similar
to SRP receptor being close to the translocon)
- TOM = translocase (or transporter) of the
outer membrane
➢ Helps to move proteins pas the OMM and
into the intermembrane space
- Different signal bind to different receptors
different proteins have different targeting
sequences have DIFFERENT TOM
COMPLEXES

Post Translational Import of IMM Proteins

- Proteins delivered to the IMS then interact
with the TIM22 complex interact with any
protein with a hydrophobic sequence
translates laterally into the IMM
- TIM = TRANSLOCASE of Inner Membrane
- Several proteins involved in energy
production are imported by this pathway

Post Translational Import of Matrix Proteins

- These proteins are directed to the TIM 23
COMPLEX found on the IMM
- Move into the matrix due to the positive
charges of the presequences works to drive
the protein out of the IMS and into the matrix
➢ The +ve charges are repelled by the proton
gradient in the IMS and pushed towards the
matrix
- Once in the matrix, mitochondrial processing peptidase (MPP) cuts off presequence
and chaperones help unfolded protein refold into mature form
if ha ge is dis upted, the o ’t e te the at i
Powerhouse
- We use 2e26 molecules of ATP daily mitochondria are the site of aerobic respiration
➢ Use oxygen to extract energy from macromolecules (preferentially glucose) and
convert to ATP
Mitochondrial DNA
- mtDNA is maternally
inherited
➢ once sperm has
fertilized egg, all sperm
mitochondria are destroyed
and only mitochondria from

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the egg is passed onto the embryo

➢ we get nDNA from mom and dad, but get all mtDNA from mom
- consumption of paternal mitochondria through autophagy = mitophagy
Mitochondrial Diseases
- can occur due to mutations in mtDNA most affect muscles or brain
➢ cardiomyopathy or muscular dystrophy
• these organs have high energy demand
- typically a group of disorders that affect oxidative phosphorylation (ETC)
➢ can be mutations in nuclear OR mitochondrial DNA
- mothers mtDNA has mutation and could be passed onto offspring and manifest in
disease (eg. Leigh syndrome)
Variation
- there is a large amount of clinical variation in the expression of MitoC diseases
➢ 2 people can inherit the exact same mutations, but there can be a huge difference in
phenotype
• eg. Brother and sister both have same mtDNA, but one is fully healthy, while
other is very sickly
- variation can result from
heteroplasmy
➢ the presence of a mixture of
wildtype and mutant mtDNA in the
same cell
- only a small subset of
mitochondria have a mutation
(typically 3/1700) depends on
chance
- diffe e t f e ue of ad
mitochondria this variation in
which oocyte is fertilized determines how bad the phenotype will be
- very small subset of things are coded in the mitoC if there is a mutation, then it can
be deadly b/c mitoC proteins serve an essential function
- itoC diseases are ’t er treata le either
3 Parent Babies
- 3 parent babies are being used to prevent mitoC disease
➢ the defective mitochondria i a other’s oo te is repla ed health ito ho dria
from an unrelated donor oocyte

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➢ third parent is unrelated

female that donate the mitochondria
Pronuclear Transfer
- take normal oocyte w/ good
mitochondria fertilize both
oocytes w/ sperm from same dad
remove pronucleus from donor and
add the pronucleus from biological
mother birth

Spindle Transfer
- take normal donor oocyte
remove spindle and chromosomes
from donor add biological mom
spindle and associated chromosomes and put in donor oocyte fertilized birth

Mitochondria Function
C6H12O6 + 6 O2 = 6CO2 + 6H2O + 38 ATP
- start with glucose (or other macromolecules like AA)
- involves several chemical reaction carried out by enzymes these reactions break
down glucose step-by-step releasing small bits of energy while activating carrier
molecules
➢ energy from carrier molecules is then used to make ATP
➢ animal cells are biased to glucose

How is ATP generated?

1. Glycolysis cytosol
2. K e ’s cycle mitochondrial matrix
3. Electron Transport Chain Inner mitochondrial membrane

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Glycolysis
- Glucose (6C) is oxidized in a set of 10 reactions (in cytosol) that are catalyzed by
specialized enzymes
- Hydrolyzed to 2 molecules of pyruvate (3C)
- Generate very small amount of ATP and breakdown a 6C sugar
- Net reaction creates: 2 Pyruvate, 2 ATP, 2 NADH, 2 H+ and 2H2O
- Pyruvate is then moved across the IMM into the matrix by a special pyruvate
transporter
➢ Moved to IMM in context of converting this to a 2C sugar
- Pyruvate is converted into Acetyl CoA (2C) by pyruvate dehydrogenase
➢ Is converted in the IMM
- lots of pathways for pyruvate to follow
- can be directly converted into oxaloacetate (in
TCA)
- can be directed to ferment into lactate oxidize
NADH to NAD+ in anaerobic conditions
➢ lactate is taken to the liver to be oxidized
- can also be used to make other macromolecules

TCA Cycle
- Acetyl CoA now feeds into a cyclic
chain of reaction known as the
tricarboxylic acid cycle (TCA)
- Acetyl Coa (2C) joined together with
Oxaloacetate (4C) to form Citrate
(6C) Citrate is then converted
back to Oxaloacetate during the TCA
cycle (b/c joined w/ new Acetyl CoA
upon more glycolysis)
- Reactions of NADH and FADH2 allow for the setup of reduced carrier molecules
- All of the cells energy providing molecules (polysaccharides, fats, proteins) are broke
down into metabolites that feed into the TCA to provide energy in the form of ATP
- Fatty acids can be converted into Acetyl Coa, same with AA
- TCA cycle relies on enzymes that cataluyze reactions these enzymes break down
carbon molecules using oxidation reactions
➢ Oxidation = removal of electrons sugars lose electrons

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➢ Reduction = addition of electrons to a molecule carrier molecules gain e- lost by

carbon molecules
• Eg. NADH and FADH2
- Net Result
➢ Citrate (6C) Oxaloacetate (4C)
• Create citrate using Acetyl-CoA and oxaloacetate redeem oxaloacetate while
reducing carrier molecules
➢ 3 NADH and 1 FADH2

ATP Generation
1. The hydrogen atoms are split into protons (H+) and e- and
the electrons are passed through the Electron Transport
Chain
➢ Make ATP by splitting H+ in H+ and e- pump H+ out to
create a gradient pumps H+ down gradient and creates
ATP
- Energy release from the ETC is used to pump the protons
from the matrix into the intermembrane space
2. The Proton Gradient across the IMM is used to make ATP
➢ Use ATP-Synthase
- Entire process is called oxidative phosphorylation
Step 1 of Oxidative Phosphorylation
➢ High energy e- associated w/ NADH and FADH2 are
transferred to a series of 4 complexes of specific e-
carriers located in the IMM
• Co ple I, II, III, a d IV // there are also 2 o ile arriers’ ubiquinone and
cytochrome c
➢ These are complexes of several
transmembrane proteins that contain
prosthetic groups (non amino acid
components tightly associated w/
complex) that serve as the e- carriers
• Prosthetic groups sit within AA
clusters eg. Iron-Sulphur cluster that
acts as an e- carrier in this case
➢ Each of these proteins gain
electrons form NADH and FADH2 and
donate them to the next carrier
• First ETC carrier becomes reduced
while NADH and FADH2 become oxidized

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- the final e- acceptor is oxygen becomes reduced

- accepts 2e- and combines w/ 2 protons from the
mitochondrial matrix to form water
- there are 3 points along the ETC where the transfer
of e- is accompanied by a release of energy I, III
and IV
➢ sets up an electrochemical gradient
- the energy released is used to pump protons from
the matrix across the IMM to the intermembrane
space
- the proteins that make up the ETC complexes are
electron carriers and proton pumps

Step 2 of Oxidative Phosphorylation

- Protons pumped out higher concentration of
[H+] in the intermembrane space compared to the matrix
➢ Have a concentration gradient
- Higher concentration of {H+} = higher positive charge in the IMS
➢ Have an electrical gradient
- Pumping protons across the IMM by the ETC components results in an electrochemical
gradient of protons
➢ A proton-motive force
ATP Synthase
- ATP synthase is a multi-protein complex that uses the proton motive force to synthesize
ATP
- Composed of 2 major complexes
1. F0 Complex
➢ Basal section embedded in the IM
➢ Contains a channel through which protons are moved from the IMS into the matrix
• Their electrochemical gradient
2. F1 Complex
➢ Is a spherical head faces the matrix
➢ Subunits are arranged like the segments of an orange
➢ Contains the catalytic sites for ATP synthesis
- Movement of H+ allows for the changing of shape and thus allows
the phosphorylating of ADP
- As H+ protons move through the � �� , the unit rotates, allowing
for the binding of ADP to Pi in the F1 subunit
- Protons move down their electrochemical gradient through the F0
subunit in (channel) in ATP synthase this movement causes a
conformational change in the ATP synthase such that the F1 and F0
subunits rotate relative to each other
➢ These rotations drive the phosphorylation of ADP into ATP

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- ATP generated in the mitochondrial matrix is moved out using ATP channels that
exchange 1 ATP for 1 ADP
➢ ATP is moved out of the matrix, ADP is brought in
• Many channels like porins allow for ATP to move out and ADP to enter
DNP
- 2,4 dinitrophenol
➢ is a hydrophobic molecule that can be
reduced w/ H+ and move through the IMM
easily
➢ essentially, energy used to create a the
gradient is wasted b/c while H+ is being pushed
out to during the ETC, DNP is bringing H+ back
in
- DNP = uncoupler
- Essentially dissipates ability of ATP-synthase
to do anything
- Un couples the 2 steps of oxidative
phosphorylation hypermetabolic state
- Proton gradient is dissipated ATP s thase does ’t ork = o ATP = fatigue
- ETC hyper works to try and compensate to replenish gradient burn calories
consume lots of oxygen (rapid breathing) chemical energy released as heat
(sweating)
Brown Fat
- Human babies have a mechanism where they mimic DNP is a controlled way
- Brown fat has tons of mitochondria
- Thermogenin is an uncoupling protein in the IMM is a proton channel
➢ Thermogenesis
- Babies have immature musculature cannot shiver thus their mitochondria have
brown fat w/ thermogenin naturally uncouples the ETC and dissipates the proton
gradients happens only in certain cells and is triggered
➢ Found in hibernating animals as well
Mitochondrial Diseases
- ETC is a substantial source of reactive oxygen species (ROS) excessive ROS can
damage macromolecules and membranes
➢ Can play a part in mitochondrial diseases
- Ma s ste s here ETC does ’t ork properl results i rea ti e o ge spe ies ei g
formed that damage those mitochondria and the membrane

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Pa ki so ’s Disease
- PINKI is a kinase of the OMM
- Parkin is an E3 Ligase
➢ Mutant parkin could result in
defective mitochondria not being
cleared onset of PD
• Essentially allows bad mitoC
to stay around eg.
Dopaminergic neurons are
sensitive to energy die if do ’t
o set of Parki so ’s
- Process: PINKI interacts w/ parkin in the OMM Parkin only interacts w/ PINK I when
there is a problem (acts as an E3 ligase to degrade) Parkin Ubs its target recruits
phagocytic proteins Mitophagy
➢ In onset of Parkinsons, this pathway does not work

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LECTURE 8 - CHLOROPLASTS

- Chloroplast have own DNA

Origin
- Originated as cyanobacteria that lived in symbiosis w/ Eukaryotic cells
➢ Phagocytosis
- Still have many similarities with cyanobacteria
➢ Similar photosynthetic machinery
• Photosynthetic membrane is identical
➢ Can divide by binary fission
• Do not know what regulates their
binary fission only know that
cytosolic GTPases are required
Structure
- Huge organelle the size of an RBC
- Outer envelope membrane
➢ Large porins
➢ Allow large molecules to get in and out
- Inner envelope membrane
➢ Highly impermeable
➢ Variety of transporters, pumps and
channels present

- Stroma – analogous to mitochondrial matrix (is similar to the mitochondrial matrix)

➢ The space w/I the chloroplast (enclosed by the inner envelope membrane)
➢ Contains many enzymes involved in carbohydrate synthesis
➢ Contains many DNA and ribosomes and protein translation factors
➢ Encodes about 100 genes
➢ 90% of chloroplast proteins are imported into the organelle
• post-translational import
- Thylakoids
➢ Grana = stacks of thylakoids
- Stroma lamellae
➢ Connect grana
• Required for ideal positioning of grana grana are equidistant from each other
to get best light energy
- Thylakoids, Grana, and Stroma Lamellae
➢ Present within the stroma
➢ Flattened membranous sacs w/ a 75 protein:25% lipid ratio (thylakoid membrane)
➢ Thylakoids are arranged in stacks called grana
➢ Space the thylakoid sacs enclose is the thylakoid lumen, space outside the sacs is
the stroma

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➢ Stroma lamellae are flattened membranes that connect thylakoids from different
grana
Importing Proteins
- Most chloroplast proteins are translated on free ribosomes in the cytosol and contain a
targeting signal that directs them to the chloroplasts
➢ Called the chloroplast transit peptide
• Located on the N-terminus
- Stroma proteins have a stroma targeting domain as part of the transit peptide
- Thylakoid proteins have a thylakoid transfer domain that is located downstream of the
stroma targeting domain
➢ Target to chloroplast then stroma (using targeting domain) then to thylakoid
(using thylakoid transfer domain)
• Eg. Thylakoid proteins have both domains
- To enter the chloroplast
➢ The protein is unfolded by Hsp70 in the cytosol
➢ The transit peptide then interacts
w/ its receptor located close to the protein
translocon channel on the outer envelope
membrane
• Called the TOC complex (translocon
of the outer membrane)
- Allows the protein to be delivered
via TOC into the intermembrane space

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- To enter the stroma

cytosol
➢ Transit peptide contains a stroma
targeting domain which interacts w/ a
second receptor that is part of the
protein translocation channel of the
Intermembrane space inner envelope membrane
• Called the TIC Complex (translocon
oF the inner membrane)
➢ Protein move into the stroma via TIC
➢ A chloroplast protease clips off the
stroma
transit peptide
• Protein is refolded by chaperones
Hsp60

Sec - To enter the Thylakoid

proteins ➢ Protein is unfolded (via Hsp70) and
gets in the IMS
➢ A stroma targeting domain of the
transit peptide interacts w/ a receptors
associated with the TIC on the inner
envelope membrane
➢ Protein is delivered to the stroma
via TIC
➢ Stromal targeting domain is cut off
by a peptidase in the stroma reveals
the thylakoid transfer domain downstream of the stroma targeting domains (is
recognized by a different receptor)
➢ This signal directs translocation into or across the thylakoid membrane process
similar to bacteria
• Sec proteins bind to something that brings signal to channel become part
of channels (similar to peroxisome)
Proteins embedded in thylakoid membrane
- These proteins are synthesized on ribosomes present w/I the stroma
➢ Dock on to the thylakoid membranes and translocate proteins
• Ribosomes move to thylakoid membrane dock translate protein into the
membrane
• Similar to ribosomes that dock at RER and translocate proteins into the ER lumen
and membrane
Mitochondrial Genome
- 37 genes, single, circular dsDNA
- UGA codes for tryptophan
Chloroplast Genome
- Same as mitoC, but 120-130 genes // some genes have been swapped to nDNA //
Mullers’s ratchet asexual to sexual

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Chloroplast Function
- Is the site of photosynthesis
➢ Process by which carbon dioxide and water are converted into glucose requires
energy
➢ Occurs in plants, eukaryotic algae, some protists, and several prokaryotes
(cyanobacteria)
Photosynthesis
- Overall Reaction: 6CO2 + 6H2O +light + ATP = 6O2 + C6H12O6
➢ Start w/ sunlight and go through a multi-step process that can be classified as:
• 1. Light dependent reactions
o light energy is absorbed and converted into chemical energy
o creates ATP and NADPH
• 2. Light Independent Reactions
o chemical energy is then used to convert carbon dioxide into
carbohydrates
Light Dependent Reaction
- conversion of light energy into chemical energy
- occurs entirely within the thylakoid
membrane
- requires the green pigment chlorophyll in
thylakoid membranes
• Pigments
➢ Compound that appear coloured because
they only absorb light of specific wavelengths
➢ Chlorophyll absorbs mainly red and blue
green is reflected back to our eyes
- About 300 chlorophyll molecules are
arranged in large pigment-protein complexes
called photosystems
➢ Each photosystem contain an antenna
complex that capture the light energy and a
reaction centre that converts the light energy
into chemical energy
• Electrons are excited and bounced around
- 2 photosystems PSI and PSII as well as
several proteins similar to those of the ETC all
play a role in light-dep rxns of photosynthesis

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Overview
- light dep. Reactions produce both ATP and NADPH
➢ nicotinamide adenine dinucleotide phosphate
➢ need 2 photons of light to do this
- 2 photons of lights are absorbed by 2 diff. photosystems that act in a series
- ATP is made after the first photon is absorbed (PSII) and NADPH is made after the 2nd
photon is absorbed (PSI)
Steps
- Light travels in the form of photons
- Chlorophyll in photosystem II (PSII) absorbs the first photon e- in Chlorophyll
become excited and move to a higher orbital
- These e- are then passed to another acceptor pigment molecules along an ETC as
they move, they drive a proton pump that pushes in H+ protons into the thylakoid
lumen
- In meantime, the second photon is absorbed by PSI e- in Chlorophyll become excited
and move to higher orbital e- are passed along in an ETC used to reduce NADP+ to
NADPH
- Activated e- from PSII moves along ETC and help establish the proton gradient ATP
synthase uses this gradient to produce ATP
- ATP synthase drives the synthesis of ATP on the stromal side of the thylakoid
membrane
Holes?
- When the e- are moving, they are leaving behind an area in the photosystem where
there are no e- present photosystem is out
of e-
- The e- from PSII travel along the ETC and fill
the e- hole of PSI allowing its reaction centre to
return to normal state
- The e- hole left behind in PSII is filled using
water
➢ Reaction centre in PSII contains a water-
splitting enzymes splits water into protons,
electrons, and oxygen
• Called the oxygen-evolving complex
➢ Removes electrons from water to replenish
loss of e- form PSII, H+ protons remain in
thylakoid lumen, oxygen is released
➢ Water-splitting enzyme is strongest
oxidizing agent known process is called
photolysis

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ATP Synthesis
- ATP synthesis in the Chloroplasts
➢ Process is called photophosphorylation
➢ Proton gradient is built up high [H+] in the thylakoid lumen ATP synthase uses
this gradient
- ATP synthase in the Chloroplasts
➢ CF0 base complex is anchored in the
thylakoid membrane has channels that
directs protons from lumen to stroma
➢ CF1 head complex is out facing the stroma
• Movement of CF1 head complex causes the
rotation of the 2 subunits relative to eachother
drive the phosphorylation of ADP to ATP
• Phosphorylation occurs in the stroma

Light Independent Reactions

- Utilization of chemical energy to reduce
carbon dioxide to glucose occurs in the
Chloroplast stroma
➢ Can also occur in the plant cytosol
- The process is called the Calvin Cycle
➢ Uses the energy form ATP and the reducing
power of NADPH (both generated form
the light indep. Reactions) to fix CO2
into organic carbon molecules
- uses up 18 ATP and 12 NADPH
molecules
- 1. Carried out by RuBisco (which
creates the unstable, carbon
intermediate)
➢ predate aerobic reactions, and is
most abundant protein on earth
➢ fixes carbon inorganic CO2
organic carbon molecules
• very slow b/c has to choose
between carbon dioxide and oxygen

Calvin Cycle
- Rubisco attaches CO2 to RuBP (5C)
to make a 6C intermediate 6C splits
into 2 molecules of PGA (3C)
- PGA is then converted into GAP (3C) using the ATP and protons from NADPH can also
be used to create sugars
- Some of the GAP is then recycled to regenerate RuBP cyclic nature

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Products
- The rest of the GAP (3C) can be
a. Exported to the cytosol and
converted into glucose or sucrose
➢ Occurs through a special
transporter; phosphate-triosephosphate
exchanger
• Exchanges 1 GAP for 1 phosphate
• Can also exchange 2 Pi for a GAP
➢ Sucrose is transported out of leaf
cells to provide energy for other parts of
the plants
b. Converted to glucose and then to
starch, which is then stored within
granules in the Chloroplasts
➢ Similar to glycogen in animals
➢ Provides plant w/ energy when light
reactions are not possible
- Plants release 450 trillion Kg of
oxygen into atmosphere, fix 500 trillion kg
of co2 into carbohydrates
Something else to think about
- Transgenic plants tomato transgenic plants are resistant
to cold or extreme heat
➢ These plants express a lot of glycine betaine (a modified
amino-acid)
• That acts as an osmolyte solute
• Is expressed a lot by chloroplasts
➢ Essentially plants show thermo-tolerance
- glyceine betaine is ampiphillic bridges the interactions
between transmembrane proteins and peripheral membrane
proteins
- essentially glues water splitting enzyme to PSII
prevents dissociation under heat stress (as the H2O splitting enzyme is a PMP)

Microalgae
- are a large and diverse group of photosynthetic organism have doubling times of 3h
- can be grown in enclosed photobioreceptors (grown in media bag that prevents their
escape into env.)
- used to make novel metabolites can manipulate their genomes eg. Gene encoding
drug we want inserted into chloroplast can be produced as a metabolite algae
produces lots of drugs as a result
- also produce ethanol (through pyruvate fermentation)

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Biofuels
- strain of cyanobacteria has been genetically engineered to enhance ethanol production
can be collected mass produced use ethanol to replace fossil fuels
Bioengineering
- chloroplast genomes altered to increase
➢ pest resistance create gene that encodes inhib. RNA molecules against essential
insect genes
➢ biopharmaceuticals plants express genes encoding insulin insulin lettuce (is
broken down in intestine and insulin goes directly into bloodstream

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LECTURE 9: MICROTUBULES

- Cytoskelton provides structure and support for cells

• Cytosol fluid matrix has water, soluble proteins (cytoskeletal proteins are
soluble), ions
➢ Cytoskeletal protei s stay i the ytosol e ause they do ’t ha e a sig al for any
other place
Proteins making up cytoskeleton
- Proteins that make up the cytoskeleton reside in the cytosol these cytoskeletal
elements are created by polymers of proteins
➢ Formed by joining several protein subunits together through weak, non-covalent
bonds (Van der Waals forces)
- Rapid assembly and disassembly of these polymers is a key characteristic required for
their function
Functions
- Provide structure and support a scaffold
- Provide an internal framework that positions organelles
• Organelles eg. Lysosomes are perinuclear (close to nucleus) brought there
and anchored by cytoskeletal elements
- Allow for the movement of materials and
organelles within cells intracellular
transport
- Required for cell motility and cell functions
phagocytosis (eg. Allows for flexibility for
things to enter the vesicles)
- Machinery for daughter cells microtubules
move DNA
Components
- 3 components:
1. Microtubules
2. Microfilaments (actin)
3. Intermediate filaments
- There are proteins that help their communication done by resistance forces

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- Peroxisomes can travel along tubulin, and are then anchored

down into a spot
Tubulin Dimers
- Hollow, tubular structures found in almost all eukaryotic cells
- 25nm in diameter
- the building blocks are made up of a protein known as tubulin
➢ tubulin subunits come in 2 forms:
➢ very similar in structure and form heterodimers similar
structure allows for easy building
- highly conserved
Protofilaments
- These alpha-beta dimers are the
building blocks of a long polymer
known as a protofilament
➢ Follows a head to tail
configuration
• Start w/ ℎ �� ℎ ��
- The protofilament is symmetric
➢ Alpha tubulin on one end, and beta tubulin on the
other end protofilament has polarity
- Alpha tubulin end = minus end and beta tubulin end =
plus end
➢ Minus end is usually anchored
Microtubules
- � �� ℎ ���−→
� ℎ � , � ���
- � �� ℎ ���−→ this GTP will slowly
hydrolyze into a GDP sometime after polymerization
➢ ℎ �� � ���
- the GAP for beta-tubulin is the BETA-TUBULIN of the
next heterodimer
- each microtubule is composed of 13 protofilament put together
➢ protofilament are aligned side-by-side in a circular pattern have a
hollow core
➢ each protofilament has the same polarity microtubules thus have
polarity
➢ held together by non-covalent interactions
• strongest interaction = between alpha-beta heterodimer, 2nd
strongest= between heterodimers, 3rd strongest = lateral, non-
covalent interactions

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Microtubules have Polarity

- the + and – end have a different structure
➢ the minus (-) end is anchored to the
centrosome (perinuclear region)
• polymerization begins from here
• referred to as the MTOC = microtubules
organizing centre
• starts with a type of tubulin called −
� �� at MTOC
• centrosome is composed of centrioles +
proteins + gamma-tubulin
- Y-tubulin ring complex Y-TURC
➢ Associates w/ other protein factors to
create a ring
➢ Ring serves as an anchor for –ve end
- Discovered MTOC by using nocadazole disassembles
microtubules that we have an anti-tubulin Ab for
➢ Washed off drug after use and looked at where
microtubules where assembled
Polymerization
- Microtubules polymerize and depolymerize at the + end
- Referred to as growth and shrinkage
➢ Can collapse indiv. Or in a small
group
➢ Typically see a branching
branching occurs b/c bonds holding protofilament together are
the weakest and are thus easiest to break
- MT can assemble and disassemble rapidly turnover
- Growth and shrinkage coexist in the cell half-life of an MT can
be as short as 10m
- Rapid growth and shrinkage is called dynamic instability
- Shrinkage is able to happen bc GDP-bound Beta-tubulin is prone
to Depolymerization
- GTP hydrolysis is usually occurring at a slower rate than the
addition of new GTP-tubulin dimers on the + end
➢ GDP-bound beta-tu uli i the iddle a ’t spo ta eously pop
out, but when GDP-form is on the growing end (+), it is very
unstable
• Thus, when GTP hydrolysis reaches the tip of the MT, the
structure will depolymerize catastrophe
• Rescue = shrinkage to growth
Summary: GTP hydrolysis is occurring in older subunits there is a GTP-cap at the top of the
MT / regio of di ers that ha e ’t ee hydrolyzed / add ore GTP tha hydrolyzed

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once GTP hydrolyzed dimers reach the tip of the MT, GTP cap disappears very unstable
depolymerization (catastrophe)
Dynamic Instability
- Stability of MT can be increased
➢ Eg. Certain binding proteins
1. Microtubules Associating Proteins (MAPs)
➢ Can affect stability typically increase stability
➢ MAPs have 2 domains: one that binds to MT and one that
extends as a filament
• Head binds to MT, tail extend away tail gets post-
translational modifications done (eg. Phosphorylation)
• Phosphorylation of MAPS control their binding ability
CANNOT bind if phosphorylated
➢ Control and shrinkage is regulated by MAPs
• If MAP is associated w/ MT, even if GTP is fully
hydrolyzed, the MT will be stable
2. Proteins that bind to the +end + end tracking proteins +TIPs
➢ +TIPs can regulate the rate of growth/shrinkage or attachment to a cellular structure
(chromosome, actin etc.)
- cells typically have both stable and unstable dynamic MT
➢ eg. Stable ones are used for organelle positioning (anchoring)
• highly associated with MAPs
➢ eg. Dynamic MT aid in cell motility
• used to help the cell move these provide a pool of dimers to help build other
ones that are required for a given time
Microtubule Motor Proteins
- these a multi-protein complexes that convert ATP to mechanical energy are ATPases
(use hydrolysis to move on MT)
➢ help to transport cargo on MT tracks
- motor proteins move unidirectionally along MT move according to MT polarity (have
affinity for 1 end or the other)
- hydrolysis of ATP provides the energy for
their movements
- composed of 2 domains
1. Domain that binds the MT
2. Other binds the cargo
➢ ATPase is in one of these domains to
hydrolyze ATP
Motor Proteins
- 2 types of microtubule motor proteins
1. Dynein – moves to the (-) end of the
microtubule retrograde (towards cell
nucleus)

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2. Kinesin moves to the (+) end of the microtubule anterograde (towards cell
periphery)

Conventional Kinesin
- + end directed motor protein
- is a tetramer protein 2 identical heavy chains, 2 identical light chains
4 structural features
1. Heads: ATPase, binds MT
2. Necks: determines the direction of movement
3. Stalk: provides flexibility or movement of heads
and tail
4. Tail: binds cargo light chains interact w/ cargo
- Adaptor proteins: tend to be integral or peripheral
membrane proteins that are part of the cargo
➢ Kinesin interacts w/ cargo using its tail uses
adaptor to tell if it has the right cargo adaptor is
usually part of the cargo itself
- kinesin moves using the ha d-over-ha d
mechanism of movement along MT
➢ single step is 8nm and is dependent on ATP
➢ the 2 heads are coordinate in their movement
cycles can move long distance on MT w/o falling off =
processive
- hydrolysis of ONE ATP allows ONE
HEAD to move
- there are several other motor
proteins in the kinesin family
➢ called Kinesin-related proteins =
KRPs
• related to Kinesin family that can
move either way neck region is key
• Kinesin 14 family neck region is
diff can move towards negative end
• Kinesin 13 family depolymerases
DO NOT move cargo, rather
depolymerize MT

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Cytoplasmic Dynein
- A very large protein complex composed of 2 Heavy Chains
(larger than kinesin)
➢ Have several intermediate and light chains
- Structure is made up of a head, stalk, stem + I & L chains
- Large head domains = ATPase, force generation
- Stalk domain = MT binding region
- Does not interact directly w/ cargo uses an adaptor
protein known as dynactin to bind cargo
➢ Dynactin complex interacts with cargo, NOT dynein itself
• No direct interaction between dynein and cargo, diff. form
kinesin which interacts using its tails

Functions
- Dynein:
➢ -ve end directed motor
➢ important for spindle positioning and
chromosome separation in mitosis
➢ moves organelles to their perinuclear position
(golgi/lysosomes)
➢ moves endosomes to cell interior
- Kinesin
➢ +ve end directed motor
➢ moves organelles such as peroxisomes and mito.C
towards the cell periphery
➢ moves secretory vesicles to cell periphery
- movement in either direction depends on the identity of the organelle will dictate
whether it interacts w/ Kin or Dyne.
➢ Is a push and pull interaction move
bidirectional until one recruits more of its
motor protein
Axonal Transport
- Transport in nerve cells is highly dependent on
the activity of motor proteins
➢ -ve end motor move cargo towards the cell
body
➢ +ve end directed motor move cargo out
towards the axon

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Microtubule: Cilia and Flagella

- hair-like motile organelles
- both cilia and flagella have essentially the
same structure both designed for cell
motility or for moving ECF
- all Euk. Cells have 1 cilium that is a
mechano-sensory organelle
- Cilia and Flagella are anchored into the PM
by the basal body beat waves

How do they move?

- Use MT

- Motor proteins do ’t tra sport,

but help move MT relative to
eachother
- Inner core of the cilium or flagellum
is known as the axoneme
- Axoneme
➢ Doublet MT
➢ 9 doublets on the outer ring
• A+B tubules come in
pairs
➢ 1 central pair
• control factory, do not
move
➢ 9 + 2 symmetry
• each MT moves w/ respect
to the other

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- see the dynein move the MT by #3, the

#3 dynein are inactivated and the #4 dynein
bring the top MT to match the bottom
➢ movement is being done w/ respect to the
2 microtubules
- also see that diff MT protrude in diff.
movements movement is done by dynein
➢ axonal dynein

- motor proteins also required for intraflagellar

transport (IFT)
- this transport is required for assembly and
maintenance of cilia and flagella
- in this flagellum, the dynein motors carry cargo
towards the basal body (- end) while kinesin motors
carry cargo to the tip of the flagellum

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Microtubules: Mitotic Spindles

- Green = MT spindles, Blue = DNA (chr.), Red =
points of attachment between MT and DNA
(TIP protein)
- (negative) end of MT at poles
- (positive) end of MT attached to DNA
- MT slowly disassemble at +ve end, cytoplasmic
dynein moves DNA to (negative) end of MTA
towards poles

Microtubule Function
- Cell structure, organelle position, vesicle/cargo
transport, cell motility, cell division
Mutant Analysis
- Muta t otor protei is ’t a le to deli er
pigment granules to destination albinism

Microtubules in Disease
- Defects in MAPs: eg. Tau protein becomes abnormally phosphorylated b/c MAPs
are essential for stabilizing MT, when Tau becomes abnormally phosphorylated, MT
depolymerizes leads to partially depolymerized MT that forms neurofibrillary tangles
results in neuronal death Alzhei er’s disease
• Hyperphosphorylated
- Cancer cells divide rapidly need rapid MT assembly and disassembly; are vital to this
process (spindle assembly and disassembly)
- Taxol is a drug that binds to MTs and prevents their disassembly (acts like a MAP and
stabilizes MT) binds to tubulin dimers and prevents their disassembly into MTs
➢ Old o e a ’t depoly erize = e o es a ’t for
• Taxol is used as a chemotherapeutic drug
• Derived from fungus on Pacific Yew

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Situs Inversus
Organ symmetry is reversed
➢ Stomach and spleen on right, liver is on left
- Defective dynein prevents beating of embryonic cilia that moves fluid (w/ growth
factors)

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LECTURE 10: MICROFILAMENTS

Actin Filaments
- 8 nm diameter filaments, much smaller than MT
- monomer is called G-actin = globular actin
- polymer is called F-actin = filamentous actin
microfilament
- F-actin polymers assemble in a double helix
- Microfilaments have polarity
➢ (-) end = point end
➢ (+) end = barbed end
- Polymerization of F-actin: See a greater
➢ Actin monomers can be added to either end propensity to fall
• When the G-actin concentration is high off, is more likely
to fall off at the
➢ Occurs preferentially at the +, barbed end, negative end
• Occurs when G-actin conc. Is low
- Depolymerization of F-actin
➢ Occurs preferentially at the (-) end or point end
➢ Actin is constantly assembling and disassembling

- As monomers are added preferentially to one end and removed

preferentially form the other end, it appears as if the monomers are moving
towards one end (- end) pointed end
➢ Called treadmilling
- Polymerization of F-actin requires ATP
➢ Actin is an ATPase
- The hydrolysis of ATP occurs sometime after polymerization (similar to
GTP hydrolysis in MT)
➢ G-actin is thus an ATPase
- Most actin filaments thus have ADP-actin subunits

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- Monomer sequestering proteins keep ATP on the G-actin ATP to preserve the G-actin

Actin Motors
- Myosins are motors that move along the
F-actin
➢ Move towards the + or barbed end
• Use ATP to drive movement
- 2 Classes of Myosin
1. Conventional Myosins eg. Myosin II
2. Unconventional Myosins atleast 17
diff. classes eg. Myosin I and V

Myosin II Structure
- Composed of
➢ 1 pair
of heavy
chains
• combine to form an intertwined chain called a coil
➢ 2 pairs of light chains
- heavy chain has a head domain
➢ ATP-hydrolysis domain
➢ Actin binding region
- Heavy chains coil together to form a tail
➢ Allows protein to form bipolar filaments
• Which are staggered arrangements of myosin
molecules such that their tails point inwards and
heads point outwards
• Tails interact w/ each other to hold MF together
• All tails are in the middle
Myosin II Function
- Present in several cell types especially muscle

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➢ Muscle cells require myosin II for muscle contraction

- In most cells, myosin II is required for cell motility and
cytokinesis (during C.D)
➢ Serves to split the cytoplasm during C.D
Muscle Contraction
- Muscle cells are very long, cylindrical cells w/ hundreds of
nuclei called muscle fibres (formed by the fusion of many
cells)
- Muscle cells are a collection of myofibril
- Myofibrils are composed of repeating structures called
sarcomeres
Sarcomeres
- Sarcomeres are made up of thick and thin filaments with
regions of overlap between them these are the
contractile units of muscles
➢ Thin filaments = microfilaments (actin)
• Stabilized and anchored F-actin filaments
➢ Thick filaments = myosin II bipolar filaments
- Give muscles a striated appearance
- banding pattern and the sets of filaments overlapping in
regions
- what type of sarcomere banding pattern and size
depends on what the muscle is doing
- actin filaments are bonded to the Z-disc, while the
bipolar filaments are in the middle
- head domains are able to touch actin move and
cause muscle contraction
- small bipolar filaments (2 myosin II molecules) can
mediate that movement of actin filaments w/ respect
to each other
➢ there is no change in length, they are just sliding
Steps: myosin heads bound actin use ATP to move actin
inwards move w/ respect to each other sarcomere
contract = muscle contracts

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- the neck region

serves as a sliding lever
movement of actin is
done by the head, which
is caused by the
movement of the neck
- thus, manipulation of
the neck domain allows
for muscular contraction

Actin Binding Proteins

- Calcium binding
proteins are bound to
thin filaments
➢ regulate the
interaction between
myosin heads and thin
filaments
➢ respond to nerve impulses intracellular
Ca2+ levels increase troponin binds to Ca2+
and shift its position along with tropomyosin
thin and thick filaments interact muscle
contracts

Unconventional Myosins
- unconventional Myosins are involved in:
intracellular transport, microvilli formation DO
NOT move filaments
- eg. Myosin V
➢ contain 2 heads like Myosin II
➢ has a very long neck region allow this myosin
to make long strides on actin
➢ has functions in intracellular transport
• head binds actin
• tail finds vesicles/cargo
• uses ATP to move to (+) end
➢ large compound compared to conventional
➢ carries cargo, does not make filaments
➢ moves using a hand over hand mechanism
- nerve cell axons are full of MT and Ifs but synapse regions have lots of actin cargo
travelling to synapse are often transferred from MT motor to Myosin V

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- nerve cell axons are full of MT and IFs but

synapse regions have lots of actin cargo
travelling to synapse are often transferred
from MT motor to Myosin V

Myosin I
- has a single head
- much smaller than other myosins
- localized in the microvilli
➢ microvilli are surface extensions of
intestinal epithelial cells
➢ structure is supported by F-actin filaments
• myosin I is thought to help stabilize this
structure attached MF to PM

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Actin Binding Proteins

- more than 100+ actin binding
proteins are involved in organizing
actin filaments in the cell
a. Nucleating Proteins
➢ Begin actin polymerization
first step involves assembly of 2-3
monomers
➢ Needed for building actin in
certain region of the cell eg. Cell
respond to stimulus by moving its PM
nucleating proteins direct
monomers to that area
• Eg. Arp 2/3
b. Monomer-sequestering
proteins
➢ Bind G-actin and stop it from polymerizing bind ATP-actin
➢ Reason for high monomer concentration in cytosol
• Eg. Thymosin
c. Capping Proteins
➢ Bind + or – end of F-actin and prevent growth or shrinkage
➢ Means to regulate length of microfilaments
d. Cross-linking Proteins
➢ Known as bundling proteins (serve as a bridge to other MF, help stabilize)
➢ Direct the organization of MF into networks
➢ Cross-link filaments to make them stable structures
➢ Useful for adding rigidity/support to internal Skelton of cell
• Eg. Fimbrin
e. Plasma Membrane Binding Proteins
➢ Bind actin filaments to PM
➢ Are typically PMP and are required for cell motility
• Eg. Spectrin protein family, dystrophin
➢ Also help give info from fluctuations in PM to interior of the cell

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Microfilament Cell Migration

- Example of non-muscle motility for cells
➢ Is essential for: development of an embryo,
formation of blood vessels, immune cell functions,
wound healing
- Is a series of repetitive actions
1. A part of the cell protrudes in the direction that the
cell wants to move called a Lamellipodium
• Lamellipodium is a piece of PM w/ actin supporting
it
➢ Cell moves in its direction
2. Cell protrusions attach to the surface beneath
ake surfa e o ta t w/ what’s e eath the
➢ Create the leading edge
3. The bulk of the cell is pulled forward towards those
surface contacts
➢ Essentially rest of cell is pulled forward and moves
above the leading edge
4. Cell breaks its rear contacts trailing edge/tail
- Lamellipodium have ruffled membranes
- the front of Lamellipodium is actin, myosin is
behind (NOT in the leading edge area)
- the cell protrusion forward and pulling forward are
generated by actin polymerization
- forces for the retraction of the rear are generated
by Myosin II motor
➢ myosin II allows for the back part of the cell to be
pulled to the leading edge

Directed Cell Motility

- a stimulus is received at one end of the cell PM
receptors bind proteins
- a family of proteins in the cytosol becomes
activated WASP proteins
- WASP proteins activate Arp2/3 complex proteins
these serve as the nucleating sites for actin
polymerization new MF form
- Activated Arp 2/3 proteins bind to new actin
filaments allow for more MF to form (branches
form) growing barbed end pushes the PM outwards in the direction of the stimulus
➢ As newer MF are formed, older ones are dissembled using proteins such as cofflin
Summary: stimulus is detected by cell binds to PM PM triggers response WASP
proteins are activated WASP activates Arp 2/3 which serve as nucleating site and allow for

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new MF formation and branching

growing MF allow the barbed end to push
outwards in the direction of the stimulus
- Cofflin distinguishes old actin from
new by the ADP binds firmly to old
actin and causes it to break apart
- ARP 2/3 pulls monomeric actin
(held by monomer sequestering protein
profilin)

MF function
- Allow for cell changes in shape
➢ Actin cytoskeleton often enriched under the PM called the cell cortex (the region
underneath the PM w/ lots of actin monomers)
- Allows for cell motility (eg. Contraction, phagocytosis)
- Structural support of cell structures (eg. Microvilli)
- Intracellular transport)

Microfilaments in Disease
- Duchenne-type Muscular Dystrophy:
➢ Lack of dystrophin
• Dystrophin anchors actin filaments to the PM in
muscle cells structural support
• Both cardiac and skeletal muscles are affected
cells lose support and eventually these muscles
deteriorate
- Dystrophin is a PM binding protein that binds actin
and provides it supports
- This is a progressive disease gets worse over
time
- Dystrophin also interacts w/ other proteins in the
cytoplasm eg. If any problem with that complex =
disease

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- Griscelli Syndrome
➢ Humans lack an unconventional myosin Myosin Va (5a)
• MyoVa is required for transport of pigment granules (melanosomes) into
pigment cells (melanocytes)
• Melanosomes are moved to the cell periphery where they are transferred to hair
follicles and are incorporated into developing hair patients with Gricelli do not
have this type of transport = albinism
- Referring to new example of Myosin V in class when MyoVa loses its phosphate, it
can move a little on the MT and get the melanosome melanophillin is req. for this
interaction
➢ Lack of melanophillin OR Rab27 can lead to Griscelli

Cardiac Muscle
- Even subtle changes in actin/myosin of heart muscle can lead to serious disease
familial cardiomyopathies
➢ Responsible for sudden death in athletes
Enteropathogenic E.Coli
- Binds to epithelial cells injects (effector proteins) proteins that ACTIVATE WASP and
Arp2/3
➢ These proteins polymerize actin which the bacteria uses as a pedestal to sit on
• Results in the disappearance of the microvilli and poor absorption of nutrients
• Lack of nutrient absorption = diarrhea
Salmonella
- Attaches to epithelial cells inject effector proteins activated WASP and Arp2/3
➢ Induced Lamellipodium formation and ruffled membranes ruffled membrane
allow for it to enter cell (endocytosed and then replicated in vesicles

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INTERMEDIATE FILAMENTS (IFS)

- Solid, unbranched, rope-like fibres 10nm
diameter thus intermediate in size to MT
and MF
- Can be linked to the other cytoskeletal
elements through bridges formed by plectin
➢ Binds one IF on one end and another IF,
actin or MT on the other end
➢ Helps to provide strength

Formation
- Have alpha-helical rod shaped monomers
spontaneously form homodimers
- Dimers have polarity N and C terminii of
the polypeptides
➢ Monomers associate in parallel N to N, C
to C
➢ the dimers associate w/ eachother in a
staggered and anti-parallel fashion to form
tetramers (that lack polarity) polymerize into
Ifs
• tetramers polymerize into
IFS
Process: spontaneously create a
homodimer have polarity
join other homodimers create
a tetramer lose polarity 8
tetramers come together
associate laterally create a unit
length of IM join w/ other unit
lengths mature IM
- IF lack polarity and are very difficult to solubilize (very stbale)
- Their polymerization ad depolymerization is controlled by
phosphorylation of various subunit proteins
➢ Unlike MF or MT, new subunits are added to the middle of IFs, not
end (unit lengths are added)
- Keratins are the main protein of skin cells new keratin is
incorporated into middle of preexisting IF
- Ifs have tissue-specific function some are more critical than others
➢ Are not used for intracellular transport
- Keratin filaments primary structure of epithelial cells
➢ Ifs in neurons = neurofilaments made up of IV group of IF
subunits
➢ IFS in muscle cells = desmin fibrils key role in muscle strength

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IFs in Disease
- Epidermolysis bullosa simplex (EBS) mutation of IF = loss of strength
➢ Skin of these patients is fragile, easy to blister
➢ Caused by mutation in keratin family in epithelial cells
• Lots of blister on weight bearing regions
- Desmin-related myopathy
➢ Mutation in Desmin (type II) mutant, aggregated IFs
➢ Skeletal and cardiac muscle do not have normal strengthened IFs
• Aggregated IFs interfere w/ MFs and MTs (eg. Desmins crosslinks w/ MT and
causes aggregations that affect MT transport)
• Patients suffer from weak muscles, often die from heart failure

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LECTURE 11 – EXTRA CELLULAR MATRIX

- The ECM is everything that surrounds the cells is made by the cells
- An organized network/meshwork of extracellular materials present beyond the PM
- ECM sits above the cell and can trap proteins to serve as signals
- Cartilage producing cells = chondrocytes its ECF is very flexible

ECM Function
- provides support to cells and tissues
(helps make contacts, help cells move)
- gi es cells ide tity
- can be site of cell attachment
- generates signals (eg. Survivial
signals, cell migration signals)
- can regulate cell function (eg.
Maintain cells in differentiated/function state ex. Mammary gland epithelial cells
produce milk only when their proper ECM is present)
Composition
- composed of a mesh of extended, fibrous proteins that are secreted by the cell
(exocytosed)
1. Collagens (most predominant)
2. Proteoglycans
3. Fibronectins
4. Laminins
➢ Diff. cells contain a diff. ratio of each
- Amount of water widely varies (eg. Blood vs bone)
- Amount of mineral present may vary (eg. High calcium phosphate in bone allows the
ECM to be very rigid)
- Gives bone its skeletal strength

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1. Collagens
- Family of secreted, fibrous glycoproteins
- Secreted by fibroblasts, osteoblasts,
smooth muscle, and epithelial cells
- Collagen is the most abundant protein in
animals has 19 genes
- Synthesized in the RER as pro-collagen
➢ Pro-collagen can form homotrimer or
heterotrimers that wind together in a triple,
rod like helix
• Provide high-tensile strength
- Pro collagen is secreted then processed
by EC enzymes mature collegen then
assembled into collagen fibrils which in turn
make collagen fibres
Process: pro-collagen is synthesized in RER
secreted by the cell post-translationally
modified by extracellular enzymes made
into collagen fibrils mature into collagen
fibres
Mutations
- Mutations in collagen can lead to severe
disease mutations in Type I collagen results in osteogenesis imperfecta
➢ Brittle-bone disease
• Fragile bones, weak tendons, thin skin
• Collagen fibrils are unable to assemble properly
- Other mutations can cause hyper flexible joints and hyper extensive skin
➢ Mutations cause defects in collagen structure
- These utatio s sho collage ’s alue it works to resist pulling forces

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2. Proteoglycans
- Proteoglycan = protein-
polysaccharide complex
= protein + Glycosaminoglycans
- Glycosaminoglycans (GAG)
➢ Polysaccharides w/ an A-B-A-B
structure while A and B are different
sugars
➢ Many contain sulphate groups w/
negative charges
• -ve charges help control H2O n
Process: present outside the cell stick
to collagen have a sugar and protein
complex sugar is a glycosaminoglycan
- hyaluronic acid can link several
proteoglycans
- proteoglycans have –ve charges can bind several cations and water molecules
➢ thus form a gel-like, porous and hydrated matrix that is resistant to crushing forces
- collagens and proteoglycans together provides strength
and resistnace to deformation (b/c resit crushing)
➢ found in cartilage
➢ bone also contains lots of collagens and proteoglycans, but
calcium phosphate make it harden

3. Fibronectin
- 2 non-identical polypeptides joined by a pair of di-
sulphide bonds
➢ each polypeptide contains several modular functional
domains (eg. Collagen binding domain)
➢ some fibronectin domains can bind the cell bind cell-
surface proteins that are connected to cytoskeleton (binds to
integrins)
➢ other fibronectin domains can bind ECM components (eg.
Collagen) thus is a good linked connect the ECM to cells

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4. Laminins
- Are an important part of the ECM of epithelial cells serve a linking function similar to
that of fibronectin
- Epithelial cells cover most of our external surfaces and line our
internal cavities
- Epithelial cells join together to form epithelial sheets
epithelium
- Epithelial cells have a top and bottom
➢ Apital (top) and basal (bottom)
• Basal side rests on a supportive network of ECM called the basal
lamina
o Basal lamina = laminins + collagen (type IV, do not make fibrils
that organize into networks)
o Network provides both strength and flexibility

Dynamic Properties of ECM

- the ECM is continually being remodeled degraded and
reconstructed
➢ this is done by enzymes known as matrix metalloprotineases-
MMPs
• have a metal ion active site that is a cofactor
- MMPs are secreted by the cell or are cell-surface proteins
break down ECM proteins and are important for wound healing (eg.
MMPs rebuild the ECM after a wound)
- Reconstruction can also be accomplished by secretion of
lysosomes (eg. Osteoclasts in bone degradation)
- Defects in MMPs (eg. If overactive) can lead to various diseases in humans eg.
Arthritis (ECM in joints becomes overtly degraded)

Cell-ECM Interactions
- Integrins
➢ Family of integral PM proteins
➢ Major ECM receptors proteins
➢ Composed of an 2 �� : �� �� come together to form a
heterodimer
• Diff. types of subunits can form diff. integrins (eg. Beta 3 and alpha 1 etc.)
Major functions of Integrins
1. Adhesion: binding to ECM or to other cells
2. Signaling: respond to signal from the inside and outside (can also send signals to
inside or out)

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1. Adhesion
- Integrins bind to a specific motif found on
several ECM proteins known as the RGD
MOTIF
➢ Arginine – Glycine – Aspartic Acid
• Acts as a bridge on the further side, is
located on the cell-binding domain that
allows for the integrin to bind to RGD
- Bind cytoskeletal proteins on the cytosolic
side and transfer any stress from the ECM
et ork to the cell’s cytosolic et ork
- Also have binding sites for cytosolic
enzymes (transmit or receive) that are
involved in sending or receiving signals
in this example, integrin would bind to the
RGD on the cell-binding domain of fibronectin
2. Signaling
- Integrins can change their conformation
dramatically in response to the binding of
protein on the extracellular side or cell interior
- The change in conformation makes them
either active or inactive
➢ Can go from a bent (folded) inactive state
to an extended, active form
- Outside-In Activation
• Bind ECM components on the extracellular
side causes cytosolic tails to binds to
various cytosolic proteins as a response
- Inside-Out Activation
• Bind to proteins on the cytosolic side
o Can also be a post-translational
modification (eg. Phosphorylation)
• Binding of proteins on cytosolic side allows
for a conformation change that allows the
integrins to bind to various ECM components

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Cell-ECM interactions: FOCAL ADHESIONS

- Focal adhesions: points of contact between the cell and
the ECM
➢ ECM is part of the substratum or surface to which the cell
is attached to
➢ Force can be seen being exerted at focal adhesion as the
protein interactions literally allow for the cell to grab on to
the surface
- Integrins are key for this interaction allow for the
growing of cells focal adhesion are the points of contact
that allow cell to bind to their surface
➢ Cell chooses points of contact
- Talin is an actin binding protein that is
associated w/ integrin
- Focal Adhesions are attached to
microfilaments

Cell-ECM Interactions: HEMIDESMOSOMES

- The tightest attachment of a cell to its ECM
is seen in regions connected cell to its
basement membrane known as
hemidesmosomes (HDs)
- HDs are regions where integrins connect
with ECM components (like Laminins)
➢ On the cytosolic side, they interact w/
proteins called plectins that form plaques
➢ Plaques links to intermediate filaments in
the cytoskeleton

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CELL – CELL INTERACTIONS

- Proteins that mediate cell-cell interactions are:
➢ Integrins
➢ Selectins
➢ Cadherins
➢ ICAMs
- Integral membrane proteins
➢ PM proteins on one cell can contact PM proteins on another cell
• Eg. Integrins on one cell can bind the others

Selectins
- Bind particular oligosaccharides on the surface of cells
their extracellular regions have a modular structure
➢ Like fibronectin
- L-selectin can be found on circulating WBCs binds to P-
selectin on endothelial cells
• Endothelial cells = cells lining the inner surface of blood
vessels

Cadherins
- Can attach overlapping
- Cadherins are
glycoproteins that join
similar cells together
➢ Have a modular
construction of their
EC domain
- Calcium ions are bound
between the different modular domains
- Cadherins are like Velcro mediate strong cell-cell adhesions
- Can interact in multiple ways

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Immunogloblin Superfamily (related to Ab)

- Several adhesion proteins belong to the IgSF
- Have modular domains on the extra cellular side
- Ig domains on the proteins interact w/ each other
eg. L1
➢ Can also interact w/ integrins on neighboring cells
• Eg. VCAM (vascular cell adhesion molecule)

CELL-CELL SPECIALIZED INTERACTIONS

1. DESMOSOMES
- Found primarily in tissues subject to mechanical
stress
- Contain cadherins (diff. domains strcutre from
classical cadherins) link cells across a small
extracellular gap
- Linked to IF network inside the cell
➢ Link the IF network between the 2 cells
- Desmosomes have 2 cadherins linking 2 cells
together
• In hemi desmosomes, have integrins

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2. ADHERINS JUNCTION
- Important link between epithelial
cells cadherins involved in these
sites
- Binds to cytosolic proteins that link
to the actin cytoskeleton
- Involved in outside-in signaling
➢ Similar to focal adhesions

3. GAP JUNCTIONS
- Specialized areas of intercellular
communication plasma membrane
comes very close together when they
form gap junctions
- Made from a family of integral
membrane proteins Connexins
- Connexins form diff. cells come
together to form a gap junction
➢ Connexin : a channel that spans the
PM of both neighboring cell non-
selective gated channels ions and
small fluorescent dyes can move
between cells via Connexins
- Cardiac muscle has lots of gap
junctions ions pass through and muscles
contract in symphony
4. TIGHT JUNCTIONS
- Act like a fence to stop the movement of
molecules in the space between
neighbouring epithelial cells
- Proteins known as Occludiins and Claudins
are required for TJs transmembrane
proteins
- The Blood-brain barrier is composed of TJs
- Stop the Para cellular pathway
- Also see example in intestinal epithelial
cells TJs stop digestive juices from entering
BS or tissues

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ECM and Inflammation

- If bacteria enter our bodies through a skin
wound WBC have to get past endothelial
cells lining the Blood. V to get the invading
bacteria have to disrupt the cells to get
into the infected tissues
➢ Inflammation immunoinfiltration
1. Chemical signals from damaged
cells, activates endothelial cells express
selectins on their surface upon activation
(secreted ligand by damaged cells allow
these cells to express)
2. Selectins on neutrophil interact w/
endothelial cell selectins
3. Neutrophil becomes activated by
signal from endothelial cells integrins on
its cell surface become activated
➢ Follows outside-in signaling
4. Activated integrins bind ICAMs on
the endothelial cells stops them from
moving entirely (cell gets docked)
5. Neutrophils disassemble adherens
junction (secrete compound that will
dissociate it) between endothelial cells
pass through the cells and infiltrate

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ECM and Pathogens

- Heliobacter Pylori
➢ Attaches to epithelial cells in stomach injects effector proteins that disrupt tight
junctions epithelial sheet has breaks in it gastric juices flow in ulcers

Tissues: Composition and Maintainence

- Tissues eg. Skin
➢ Composed of a complex micture of cell types
➢ Each cell in the tissue must maintain its own
identity
➢ Each cell must coexist in the same env. As the
other cell types
- Keratinocytes makes Kereatin (IFs)
- Melanocytes make melanin level of
melanocyte activity varies between different people
➢ Less hair colour = less melanocyte activity
- Macrophages destroy damaged/dying cells
- Lymphocytes fight infection
- Endothelial cells line the blood vessls that carry
oxygen, and remove waste
- Fibroblasts make ECM

Tissue Maintainence and Removal

- Cells in various tissues vary enourmously in their
rate and pattern of turnover dying cells are
replaced
- Nerve cells may last a lifetime, intestinal cells
replaced every few days
- Many cells do not divide differentiated RBCs
➢ To make more of these differentiated cells, need
dividing precursor cells
• These cells are derived from stem cells in the
tissues stem cells present in small numbers in the
tissues a d are specialized for the tissues they’re i
• Can differentiate into diff. cell types and can self-
renew
- Use hematopoetic stem cells

CELL DIFFERENTIATION
- The process by which a cell become specialized
achieved by changes in gene expression (all contain
the same genetic material)
➢ Diff. cell types have really specialized functions

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- Intestinal epithelial cells: secretion of digestive enzymes, absorb digested contents,

keep juices away from BS
➢ Cell Specializations
• 1. Secretion Machinery (digestive enzyme secretory vesicles, RER, MTs +
kinesin)
• 2. Microvilli actin, myosin I
• 3. Tight Junctions upregulate occuludin and claudins
• 4. Desmosomes cadherins and IFs for strength (cell-ECM interactions)
• 5. Hemidesmosomes integrin attachment to basal lamina (w/ IFs and plectin)
- Hepatocytes: produce bile protein detoxify bloodstream
➢ Cell specialziations
1. Secretion machinery bile, ER, MTs + Kinesin
2. Lots of SER
- Cardiac Muscle cell: synchronized contractions to propel blood through heart
➢ Cell specialziations
1. Desmosomes strength
2. Gap Junctions allow for synchoronic ion movements
3. Contractile Machinery F-actin and Myosin
4. Mitochondria need lots of ATP
5. Sarcomere needs lots of calcium extensive RER
• Has the sarcoplasmic reticulum (calcium stores)
- Neurons: early in dev. send our migratory axons towards grow cones synapses
➢ After differnetiaiton get nerve impulses formation of secretory vesicles
➢ Have a lot of directed cell motility
Specializations
1. Cell Migration Machinery actin/WASP/Arp2/3/Integrins
2. Nerve Impulses sodium + potassium channels
3. Secretion machinery lots of ER, MTs (and MAPS), kinesins, neurofilaments, actni,
MyosinVa
4. Mitochondria need lots of ATP

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LECTURE 12: STUDYING PROTEINS II

Step I Protein Purification

1. What is the primary sequence?

- Protein purified from cell extract cut out

of a 2-D gel
- Protein is broken into smaller pieces use
protease like trypsin
➢ Is very specific cuts certain areas a
certain # of times has a high affinity for
glycine
- These peptides are dies onto a metal plate
and heated w/ a laser allow for peptide to
become in gas phase
➢ Try to use sample have lots of e- (from the metal plate) try
to knock off the electrons goal is to create positively charged
peptides
- Are fired from the plate as a gas through a magnetic field within a
mass spectrometer (placed facing towards a negative plate)
Mass Spectrometry
- Peptide ions separate from each other based on their mass and
charge smaller ions travel fast, highly charged ones also travel
faster
- Separate according to their mass and charge ratio m/z
- Ions hit a detector on one end which plots the signatures of the
peptides based on the time it
takes to reach the plates
protein is then identified from
a sequence database
- Wo ’t ha e a y peptides
w/ more than a 1+ charge
if all charges are same, then
we are separating by mass
- Protein is cut up put in
mass spectrometer in gas
phase for m/z ratio
database
- Use the unique masses
measured for the peptides to determine the
amino acid sequences
2. What is the tertiary structure of the protein?

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- Start w/ lots of purified protein

remove water to make crystals of the
proteins
➢ Crystals = highly ordered arrays of
the protein (make a protein precipitate)
- Direct X-rays at it e- of the atoms
in the protein crystal will scatter the X-rays
= diffraction
➢ X-rays have very small wavelengths
and can therefore hit single atoms
- The diffraction depends on the
position of the atoms, which is determined
by the tertiary structure
- The scattered X-rays are picked up by the detector
placed behind the protein crystal = diffraction
pattern
- The position and intensity of each spot in the
diffraction pattern corresponds to the location of an
atom in the protein crystal computers analyze this
data and mathematically predict the relative spatial
positions of the atoms
- Is then combined w/ the primary sequence to
generate a protein model structure

Cryo EM

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GFP-fusion Proteins: FRET

- GFP variants can be used to detect the

distance between diff. parts of a protein or
different proteins that are a part of a complex
➢ Method is called: Fluorescence Resonance
Energy Transfer FRET
• Based on the fact that excitation energy can
be transferred form one fluorophore to another
as long as they are close together
• Process: initial wavelength excites the
donor is acceptor is nearby, it absorbs the
energy E. of acceptor causes acceptor to
fluoresce
• Fluorophores are GFP-derivatives eg. YFP-
GFP
- Protei s do ’t al ays ha e the sa e shape this technique is used in conjunction w.
X-ray crystallography/Cryo EM

3. Where is the protein localized?

- Make GFP-fusion protein
- Immunofluorescence

4. Protein Function?
1. Look for Interacting partners (you are who your friends are)
2. Use loss of function analysis
➢ Remove a gene that encodes protein (gene knock-out)
➢ Remove mRNA that encodes the protein (siRNA)

Co-Immunoprecipitation assays are

used for this purpose
- General principle
➢ Within a cell, proteins A, B, and C
interact together
• If purify A and have a good Ab
against A, then you co- pull out proteins
B and C along with A, using the anti-A
Ab
• Called co-immunoprecipitation
- Add a bead that clumps w/ the
protein create a giant, heavy complex sinks to bottom o A a d other
proteins interact w/ it

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- Can also use magnetic beats as an easy means of

separating the Ab-bound proteins form the unbound
components

TAP-Tagging
- A global scale protein-protein network can be creating
➢ The gene encoding protein of interest is fused to a gene
encoding a TAP tag
• TAP tag can be purified easily using affinity
chromatography
- Fusion gene protein expressed in cells
- Co-IP all interacting partner of protein of interest by using
TAP tag Ab
- All co-IP’d protei s are ide tified usi g ass-spectrometry
- TAP = tandem affinity purification

- middle is the protein

cutting site have a
Calmodulin binding site
for 2nd beads
Process: protein of
interest has TAP tag
go through column w/
Igg beads cut off IgG
portion using protease
opens Calmodulin
binding site binds to
calmodulin beads
wash with salt to wash
off sticky proteins
get proper binding
partners

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Gene-knock out
- remove the gene that encodes for
protein of interest in embryonic stem
cells introduce into embryos
(blastocysts)
- look at cells from the mice or
lookout mice behaviour/anatomy of
knockout mice

- selection marker is place on a gene

that is very similar lines up w. gene
and replaces gene of interest on the
chromosomes
- electroporan is used to introduce
DNA into the cell
- the cell is heterozygote will need
to create 2 to make a homozygote

siRNA technology:
- contain the complimentary sequence of mRNA of choice
- these small RNAs bind to the corresponding mRNA in the cell the mRNA that encodes
for the protein of interest
- transfect siRNA into the cell/organism that normally expresses the protein when
siRNA bound to mRNA create double-stranded RNA
➢ dsRNA is seen as foreign to the cell (viruses have dsRNA) is subsequently
destroyed by the cell
• no mRNA = no proteins look for changes in the organism
- have lots of siRNA b/c once lose siRNA, then protein is normally expressed again

Clinical Applications of Protein Techniques

- Mass Spectrometry: use to find unique proteins in disease cells, used to test for
drugs/doping
- Co-IPs: used to find unique protein:protein interactions in disease cells, drug
development, see what ou protein binds to
- siRNA: to down-regulate disease-causing proteins
➢ put siRNA into delivery system
a) use a liposome that can penetrate PM and release siRNA into cytosol
• is a lipid capsule that associates with the PM and release siRNA into the cell
b) Adenovirus uses its viral vectors
• Re o e ad, irule t RNA a d protei s fro iruses a d pa kage your siRNA
inside virus will inject siRNA into the cytosol
- siRNA used for survivin inhibition

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