BIOL 266 – CELL BIOLOGY

Lecture 9:
Protein sorting to organelles (II) –
secretory pathway
 nucleus Proteins are sorted from the ER onward
and from one compartment of the
endomembrane system to another
Proteins are transported by transport
vesicles
Transport vesicles become loaded with a
chloroplast
cargo of proteins from the interior space
(lumen) of one compartment, as they pinch
mitochondrion off from its membrane
These transport vesicles subsequently
peroxisome
ER discharge their cargo into a second
lysosome compartment by fusing with its membrane
Golgi
apparatus
3 TRANSPORT BY VESICLES
Proteins remain folded during the transport

Pinches off Fuses

Folded protein Transport vesicle
 A major outward pathway of vesicular transport (traffic) –
the secretory pathway

IMPORT

lysosome

Golgi apparatus
ER plasma membrane
external
medium

Secretory pathway: Proteins are transported from the ER, through the Golgi
apparatus, to the lysosome, plasma membrane or external medium
 Two branches of the secretory pathway:
(1) lysosomal branch
(2) exocytic branch (aka exocytosis)
(1) lysosomal branch
IMPORT lysosome

Golgi apparatus
ER (2) exocytosis
Each of the membrane-bound compartments of the
endomembrane system communicates with one another
by means of transport vesicles
Different types of transport vesicles:

IMPORT lysosome

Golgi apparatus
ER
 To perform its function correctly, each transport vesicle that buds
off from a compartment must:
(1) take with it only the proteins appropriate to its destination
(2) fuse only with the appropriate target membrane

IMPORT lysosome

Golgi apparatus
ER
1st example: A vesicle carrying cargo proteins
from the ER to the Golgi apparatus must:
(1) exclude protein(s) that are to stay in the ER
(2) take with it only proteins appropriate to the Golgi
(3) fuse only with with the Golgi apparatus membrane

IMPORT lysosome
2nd example: A vesicle carrying cargo protein(s) from the
trans Golgi network to the lysosome must:
(1) exclude protein(s) that are to stay in the Golgi
(2) exclude proteins that are to be transported to the plasma
membrane and external medium
(3) take with it only protein(s) appropriate to the lysosome
(4) fuse only with with the lysosomal membrane

IMPORT
lysosome

trans Golgi network

Due to this selectivity of the processes of vesicle packaging, budding and
fusion:
Each organelle of the endomembrane system maintains its own distinct
identity, that is, its own distinctive protein composition
ER: contains only protein Golgi: contains only protein
Lysosome: contains only Plasma membrane: contains only

IMPORT
lysosome

trans Golgi network

 Newly synthesized proteins destined to reside in organelles of the
endomembrane system are co-translationally imported to the rough
ER as unfolded, monomeric and unmodified polypeptides
Their folding, assembly into multimeric complexes and covalent
modification occur in the rough ER

Co-translational
import

SRP

Translocon
SRP receptor
ER
Loop of the ribosome-bound polypeptide chain
Newly synthesized proteins in the membrane and lumen of the
ER undergo five principal modifications before they reach their
final destinations, various organelles of the endomembrane
system:
(1) formation of disulfide bonds SH HS S S

(2) addition and processing of carbohydrates

(3) specific proteolytic cleavage

(4) proper folding

(5) assembly into multimeric proteins + +

Disulfide bonds are formed in the lumen of the rough ER but not in the cytosol
Cystein Cystein

Sulfhydryl Interchain
CH2 groups CH2 disulfide
bond
SH S
oxidants + enzyme

SH reductants S

CH2 CH2

CH2 CH2

SH S
Intrachain
disulfide
SH S bond
CH2 CH2

CYTOSOL: reducing environment, ER: oxidative environment,

high level of glutathione GSH low level of glutathione GSH
 Disulfide bonds are found only in secretory proteins and in
the extracellular domains of plasma membrane proteins

Function of disulfide bonds formation in the ER:

Help to stabilize the structure of those proteins that may
encounter changes in pH and degradative enzymes after
they are incorporated into the plasma membrane

Extracellular
CELL medium

pH pH can be
homeostasis changed (no pH
SH HS S S homeostasis)
SENSITIVE to RESISTANT to
changes in pH changes in pH Proteases are not
Proteases are
and to proteases and to proteases surrounded by
inside
membranes
lysosomes
secretory
extracellular domains proteins
of PM proteins
Most plasma membrane and secretory proteins that initially enter the
ER lumen or ER membrane contain one or more carbohydrate chains
The covalent attachment of short carbohydrate chains converts these
proteins into glycoproteins
This process of glycosylation is carried out by glycosylating enzymes
found in the ER but not in the cytosol

Glycosylases Glycoprotein
(oligosaccharide Glucose
protein transferases)
Galactose
Mannose
CYTOSOL: ER: several
glycosylases are oligosaccharide-
not found and protein-
specific
glycosylases are
present
 N-linked protein glycosylation in the ER
a specialized
N-acetylglucosamine mannose glucose dolichol membrane lipid

Translocon
Cytosol

dolichol
dolichol

e
gin

ER lumen
a

P
ar

Asparagine
p

Loop of the
As

P ribosome-bound P
NH2 growing
N
polypeptide chain

Oligosaccharide protein transferase

As soon as a polypeptide chain enters the ER lumen, it is
glycosylated by covalent attachment of a preformed
branched oligosaccharide to particular asparagines in
the polypeptide
 Functions of oligosaccharides covalently attached to proteins in the ER
(1) protect the protein from degradation:

Sensitive to proteases in Resistant to proteases in

the extracellular medium the extracellular medium

(2) retain the protein in the ER until it properly folded:

Unfolded or misfolded protein:
unable to exit the ER, binds to
oligosaccharide-specific chaperone + chaperone
Properly folded protein:
able to exit the ER

(3) help guide the protein to the appropriate organelle by serving as a

transport signal for packaging the protein into appropriate transport vesicles

ER ER vesicle
Correct folding of newly made proteins is facilitated by several ER
chaperones, proteins that accelerate the folding within the ER lumen

STEP 5
The ER chaperone protein Hsp70 uses
release of the ribosome
the energy of ATP hydrolysis:
(1) to pull the protein inside, into the
ER lumen
(2) to assist in the final folding of the
translocated protein into its mature,
active state

Pulling Hsp70 ATP

inside ADP Hsp70 ATP

ADP
Folding
 Folding and assembly of proteins into multimeric protein complexes
is assisted by two ER chaperones, Calnexin and Calreticulin

Calreticulin

1st step: 2nd step: 3rd step:

Protein glycosylation (1) binding to Calnexin (1) release of the
and Calreticulin ribosome
Calnexin
(2) formation of three (2) assembly of a
Protein: Hemagglutinin disulfide bonds trimeric protein
 1st mechanism that controls protein exit from the ER: ER-resident
proteins are retained in the ER by an ER retention signal

budding
ER transport
vesicle

ER retention signal: a Proteins that are able to exit from the ER:
carboxyl-terminal
tetrapeptide KDEL = (1) do not contain the ER retention signal
Lys-Asp-Glu-Leu (2) properly folded

KDEL receptor: (3) properly assembled into multisubunit

protein complexes
(1) binds to the ER retention signal
(2) acts to retain ER-resident proteins in the ER
(3) acts to retrieve proteins with the KDEL sequence
that have escaped to the cis-Golgi network and return
them to the ER
 2nd mechanism that controls protein exit from the ER:
Quality control in the ER

ER
2 1 budding
transport
vesicle
chaperone
export

Properly folded monomeric proteins and

degradation properly assembled non-resident
multimeric protein complexes are able to
exit from the ER
Misfolded or unassembled non-resident proteins: Are actively
retained in the ER lumen by binding to chaperones

2 1
Proteins that fail to fold or assemble properly: Interaction with chaperones holds
(1) are exported from the ER to the cytosol misfolded and unassembled proteins in the
(2) are then degraded in the cytosol ER until proper folding and assembly occur
 3rd mechanism that controls protein exit from the ER: the assembly
of a protein coat on the cytosolic surface of transport vesicles

budding budding
vesicle vesicle
ER ER

cytosolic coat proteins

protein budding
Vesicles that bud from the ER and other coat
membranes of organelles of the endomembrane
system have a distinct protein coat on their coated
cytosolic surface and therefore called coated vesicle
vesicles

After budding is completed, the coat is lost, uncoating

allowing the membrane of the vesicle (vesicular
membrane) to interact directly with the
membrane to which it will fuse (target budding
vesicle
membrane)
Two functions of protein coat on the cytosolic surface of budding vesicles:
(1) it shapes the donor membrane into a bud
(2) it helps to capture only cargo proteins (membrane-bound and soluble)
into budding vesicles
membrane cargo protein
ER-resident protein
KDEL receptor

budding
vesicle
ER (donor
membrane)

soluble cargo protein

Components that participate in budding of coated vesicles

small GTP-
1st step:
binding protein
Budding is initiated by
soluble cargo recruitment of a small
protein GTP-binding protein
from the cytosol to the
cytosolic surface of a
donor membrane
membrane
cargo protein
This small GTP-
binding protein
donor
membrane regulates the rate of
vesicle formation
Components that participate in budding of coated vesicles

Adapter for the

membrane cargo protein

Adapter for the soluble

cargo protein

2nd step:
Coat and adapter
proteins are recruited
from the cytosol to the
cytosolic surface of the
budding vesicles
coat protein
 Components that participate in budding of coated vesicles
Signal sequence that is recognized by an
adapter for the membrane cargo protein

3rd step:
Adapter proteins select which
membrane and soluble proteins
will enter the transport vesicles
as cargo proteins

These cargo proteins contain

short signal sequences that are
recognized by the corresponding
Signal sequence that is recognized by an
adapter for the soluble cargo protein adapters and direct them to a
specific type of transport vesicle
Components that participate in budding of coated vesicles

polymerized coat subunit proteins

4th step:
The coat subunit proteins
polymerize around the
cytosolic face of the
budding vesicle, thereby
helping the vesicle to pinch
off from the donor (parent)
organelle
 Components that participate in budding of coated vesicles

GTP

GDP
5th (final) step:
Small GTP-binding proteins hydrolyze
their binding GTP and pinch off the vesicle
 Three types of coated vesicles transport
proteins from organelle to organelle

Vesicle Transport step

COPI Vesicular transport between Golgi cisternae

Golgi ER

COPII ER Golgi

Clathrin Golgi Lysosome

 Fusion of all three types of transport vesicles with their
target membranes exhibits several common features:
(1) fusion occurs only after the coats have depolymeraized

protein budding
coat

coated uncoating uncoated

vesicle vesicle

fusion with target membrane

target membrane
Fusion of all three types of transport vesicles with their
target membranes exhibits several common features:

(2) the transport vesicle must specifically recognize the

correct transport membrane

(3) the vesicle and target membranes must fuse, thereby

delivering the contents of the vesicle to the target organelle
Fusion of all three types of transport vesicles with their
target membranes involves a conserved set of proteins:

NSF = N-ethylmaleimide sensitive factor, ATPase

SNAPs = soluble NSF attachment proteins
v-SNARE = SNAP receptor on the transport vesicle
t-SNARE = SNAP receptor on the target membrane
Rab proteins = a family of small GTP-binding proteins
 Targeting and fusion of transport vesicles with their target membranes

vesicle

Rab - GTP Rab - GTP

v-SNARE
target t-SNARE
membrane
1st step – Docking:
The attachment of a transport
vesicle to its target membrane
is mediated by binding between
vesicle v-SNAREs and t-SNAREs on
Rab - GTP Rab - GTP
the vesicle and target
membrane, respectively
Rab GTP-binding proteins are
required to facilitate formation
of v-SNARE/t-SNARE
complexes
Targeting and fusion of transport vesicles with their target membranes

vesicle

Rab - GTP Rab - GTP 2nd step – Fusion:

Fusion of the two membranes
requires GTP hydrolysis by
small Rab proteins

Rab - GDP
The NSF/SNAP complex is
Rab - GDP
recruited from the cytosol to
the membrane

SNAPs
NSF
Targeting and fusion of transport vesicles with their target membranes

Rab - GDP Rab - GDP 3rd step – Disassembly of

ATP SNARE complexes:

ADP The NSF/SNAP complex uses

the energy of ATP hydrolysis
by NSF to disassemble the v-
SNARE/t-SNARE complex,
thereby allowing the SNAREs to
be reutilized for subsequent
rounds of vesicle transport