NPTEL Biotechnology Cell Biology

Module 3 lecture 1
Transport across cell membrane
All cells are generally separated from their surrounding environment by plasma
membrane. In addition, the eukaryotic cells are compartmentalized by intracellular
membranes

that

form

the

boundaries

and

internal

structures

of

various

organelles.Thesebiological membranes are semi-permeable in nature that is their

permeability properties ensure that the specific molecules and ions readily enter the cell
and the waste products leave the cell. These movements of solutes into the cell are
mediated through the action of specific transport proteinsthat arepresent on the cell
+

membrane. Such proteins are therefore required for movements of ions, such as Na , K ,
+

Ca2 , and Cl , as well as metabolites such as pyruvate, amino acids, sugars, and
nucleotides, and even water. Transport proteins are also responsible for biological
electrochemical phenomena such as neurotransmission.
Cell membranearchitecture in transport across cell membrane:
The cell membrane plays an important role in transport of molecules. Because it acts as a
semi-permeable barrier, allowing specific molecules to cross while fencing the majority
of organically produced chemicals inside the cell. Electron microscopic examinations of
cell membranes reveal the development of the lipid bilayer model (fluid-mosaic model).
The model consists ofphospholipid, which has a polar (hydrophilic) head and two nonpolar (hydrophobic) tails. These phospholipids are aligned tail to tail so the non-polar
areas form a hydrophobic region between the hydrophilic heads on the inner and outer
surfaces of the membrane.
Permeability of molecules across phospholipid bilayer:
Most of the molecule will diffuse across a protein-free lipid bilayer down its
concentration gradient, if provided enough time. The diffusion rate is the function of the
size of the molecule and its relative solubility in oil. In general, the smaller the molecule
and the more soluble in oil (the more hydrophobic or non-polar), the more rapidly it will
diffuse across a cell membrane. Small non-polar molecules, such as O2 and CO2, readily
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dissolve in cell membrane and therefore diffuse rapidly across them whereas small
uncharged polarmolecules, such as water or urea, also diffuse across a bilayer, but much
more slowly but ethanol diffuses readily. Conclusively it can be said that lipid bilayers
are highly impermeable to charged molecules (ions) by considering its size also because
the charge and high degree of hydration of such molecules prevents them from entering
9

the hydrocarbon phase of the bilayer. Thus, these bilayers are 10 times more permeable
+

to water than to even such small ions as Na or K (M. Lodish et al., 2003).

Figure 1:Relative permeability of a pure phospholipid bilayer to various molecules. A bilayer is permeable to small
hydrophobic molecules and small uncharged polar molecules, slightly permeable to water and urea, and essentially
impermeable to ions and to large polar molecules.

Thermodynamics of transport :
The diffusion of a substance A, across the two sides of a membrane thermodynamically
resembles a chemical equilibration.
A(out) A(in)
In the following sections, the free energy of a soluteA, varies with its concentration:
,

=
=
( )
where
GAis the chemical potential(partial molar free energy) of A (the bar indicates quantity per
mole)

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G`A is the chemical potential of its standard state.

Thus, a difference arises in the concentrations of the substance on two sides of a
membrane and generates a chemical potential difference:

= ( )

( )=

[ ]

[ ]

If the concentration of A outside the membrane is greater than that inside, GA for the
transfer of A from outside to inside will be negative and the spontaneous net flow of A
will be inward. Conversely, if [A] is greater inside than outside, GA is positive and an
inward net flow of A can occur only if an exergonic process, such as ATP hydrolysis, is
coupled to it to make the overall free energy change.
The transmembrane movement of ions also depends in charge differences across the
membrane, thereby generating an electrical potential difference which is given by:
A=A(in) A(out),
whereAis termed the membrane potential.Consequently, if A is ionic, must be amended
to include the electricalwork required to transfer a mole of A across the membrane from
outside to inside:
=

[ ]

[ ]

where
ZA is the ionic charge of A
F, the Faraday constant, is the charge of a mole of electrons (96,485 C /mol; C is the
symbol for coulomb)
GA is now termed the electrochemical potentialof A.
The membrane potentials of living cells are commonly as high as 100 mV (note that 1 V
= 1 J /C).

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Types of transport process:

Two types of transport processoccuracross the membrane.
1. Non-mediated transport
2. Mediated transport
Non-mediated transport occurs through the simple diffusion process andthe driving force
for the transport of a substance through a medium depends on its chemical potential
gradient. Whereasmediated transportrequires specific carrier proteins. Thus, the substance
diffuses in the direction that eliminates its concentration gradient; at a rate proportional to
the magnitude of this gradient and also depends on its solubility in the membranes nonpolar core. Mediated transport is classified into two categories depending on the
thermodynamics of the system:
1. Passive-mediated transport, or facilitated diffusion:In this typeof process a specific
molecule flows from high concentration to low concentration.
2. Active transport:In this typeof process a specific molecule is transported from low
concentration to high concentration, that is, against its concentration gradient. Such an
endergonic process must be coupled to a sufficiently exergonic process to make it
favorable (G <0).

Figure 2: Mediated transport. (A) Passive transport and (B) Active transport

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Passive mediated transport:

Substances that are too large or polar diffuse across the lipid bilayer on their own through
membrane proteins called carriers, permeases, channels and transporters. Unlike active
transport, this process does not involve chemical energy. So the passive mediated
transport is totally dependent upon the permeability nature of cell membrane, which in
turn, is function of organization and characteristics of membrane lipids and proteins.
Types of passive transport:
1. Diffusion:
The process of the net movement of solutes from a region of high concentration to a
region of low concentration is known as diffusion. The differences of concentration
between the two regions are termed as concentration gradient and the diffusion continues
till the gradient has been vanished. Diffusion occurs down the concentration gradient.

Figure 3: Diffusion.Extracellular space contains high concentration of solutes than intracellular space and hence the solutes move
from extracellular space to intracellular space till there is no concentration gradient between the spaces.

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2. Facilitated diffusion :
The process of the movement of molecules across the cell membrane via special transport
proteins that are embedded within the cellular membrane is known as facilitated diffusion
or called carrier-mediated diffusion. Many large molecules, such as glucose, are insoluble
in lipids and too large to fit into the porins, therefore, it will bind with its specific carrier
proteins, and the complex will then be bonded to a receptor site and moved through the
cellular membrane.

Figure 4: Facilitated transport.Movement of the solutes from extracellular space to intracellular space via carrier proteins and
down its concentration gradient.

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3. Filtration:
Filtration is the process of the movement of water and solute molecules across the cell
membrane due to hydrostatic pressure generated by the system. Depending on the size of
the membrane pores, only solutes of a certain size may pass through it. The membrane
pores of the Bowman's capsule in the kidneys are very small, and only albumins (smallest
of the proteins)can filter through. On the other hand, the membrane pores of liver cells
are extremely large, to allow a variety of solutes to pass through and be metabolized.

Figure 5: Filtration

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4. Osmosis:
Osmosis is the type of diffusion of water molecules across a semi- permeable
membrane, from a solution of high water potential to a region of low water potential.
A cell with a less negative water potential will draw in water but this depends on
other factors as well such as solute potential (pressure in the cell e.g. solute
molecules) and pressure potential (external pressure e.g. cell wall).

Figure 6: Osmosis.(A) In hypertonic solution, there are more solute molecules outside the cell, which causes the water to be
sucked in that direction which leads to the shrinkage of cells. (B) In isotonic solution, there is equal concentration of solute on
both sides, henceforth the water with move back in forth. (C) In hypotonic solution, there are less solute molecules outside the
cell, since salt sucks and water will move inside the cell. The cell will gain water and grow larger, and finally burst.

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Active transport:
Active transport is the movement of a substance against its concentration gradient (i.e.
from low to high concentration). It is an endergonic process that, in most cases, is
coupled to the hydrolysis of ATP.
Types of active transport:
1. Primary active transport:Primary active transport, also called direct active
transport, directly uses energy to transport molecules across a membrane.
Example:Sodium-potassium pump, which helps to maintain the cell potential.

Figure 7: Primary active transport.The action of the sodium-potassium pump is an example of primary active transport.

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2. Secondary active transport:Secondary active transport or co-transport, also uses

energy to transport molecules across a membrane; however, in contrast to primary
active transport, there is no direct coupling of ATP; instead, the electrochemical
potential difference created by pumping ions out of the cell is instrumental.

Figure 8: Secondary active transport

The two main forms of active transport are antiport and symport.
(a) Antiport:
In antiport two species of ion or solutes are pumped in opposite directions across a
membrane. One of these species is allowed to flow from high to low concentration which
yields the entropic energy to drive the transport of the other solute from a low
concentration region to a high one.Example: the sodium-calcium exchanger or antiporter,
which allows three sodium ions into the cell to transport one calcium out.
(b) Symport:
Symport uses the downhill movement of one solute species from high to low
concentration to move another molecule uphill from low concentration to high
concentration (against its electrochemical gradient).
Example: glucose symporterSGLT1, which co-transports one glucose (or galactose)
molecule into the cell for every two sodium ions it imports into the cell.

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Examples:
+

(A) (Na K )ATPase

+

(Na - K )ATPaseactive transport system is commonly found in the plasma membranes

of higher eukaryotes, which was first characterized by Jens Skou. This transmembrane
protein consists of two types of subunits: a 110-kD non-glycosylated - subunit that
contains the enzymes catalytic activity and ion-binding sites, and a 55-kD glycoprotein
-subunit of unknown function. Sequence analysis suggests that the - subunit has eight
transmembrane -helical segments and two large cytoplasmic domains. The - subunit
has a single transmembrane helix and a large extracellular domain. The protein may
function as an ()2 tetramer in vivo.

Figure 9: (Na+ - K+) ATPase.This diagram shows the transporters dimeric structure and its orientation in the plasma
membrane. Cardiotonic steroids bind to the external surface of the transporter, thereby inhibiting transport.

The (Na K )ATPase is also called as the (Na K ) pumpbecause it pumps 3 Na out
+

of and 2 K into the cell in presence of hydrolysis of intracellular ATP. The overall
stoichiometry of the reaction is:
+

3 Na (in) + 2 K (out) + ATP + H2O 3 Na (out) + 2 K (in) + ADP + Pi

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(B) Ion GradientDriven Active Transport

For example, cells of the intestinal epithelium take up dietary glucose by Na

dependentsymport. This process is an example of secondary active transport because Na

+

gradient in these cells is maintained by the (Na K )ATPase.The Na glucose

transport system concentrates glucose inside the cell. Glucose is then transported into the
capillaries through a passive-mediated glucose uniport (which resembles GLUT1).

Figure 10: Glucose transport across Interstinal epithelium.The brushlike villi lining the small interstine greatly increases the
surface area (a), thereby facilating the absorption of the nutrients. The brush border cells from which the villi are formed (b)
concentrate glucose from the interstinal lumen in symport to Na+ (c), a process that is driven by (Na+ - K+ ) ATPase, which
is located on the capillary side of the cell and functions to maintain a low internal [Na+]. The glucose is exported to the
bloodstream via a passive-mediated uniprot system similar to GLUT1.

Differentiating mediated and non-mediated transport:

Glucose and many other compounds can enter cells by a non-mediated pathway; that is,
they slowly diffuse into cells at a rate proportional to their membrane solubility and their
concentrations on either side of the membrane. The flux(rate of transport per unit area) of
asubstance across the membrane increases with the magnitude of its concentration
gradient. If glucose moves across a membrane by means of a transport protein, its flux is
no longer linear.

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This is one of four characteristics that distinguish mediated from non-mediated transport:
1. Speed and specificity-The solubilities of the chemically similar sugars D-glucose
and D-mannitol in a synthetic lipid bilayer are similar. However, the rate at which
glucose moves through the erythrocyte membrane is four orders of magnitude
faster than that of D-mannitol. The erythrocyte membrane therefore contains a
system that transports glucose and that can distinguish D-glucose from Dmannitol.
2. Saturation-The rate of glucose transport into an erythrocyte does not increase
infinitely as the external glucose concentration increases. Such an observation is
evidence that a specific number of sites on the membrane are involved in the
transport of glucose; which becomes saturatedat high [glucose] and the plot of
glucose flux versus [glucose] is hyperbolic. The non-mediated glucose flux
increases linearly with [glucose].
3.Competition-The curve is shifted to the right in the presence of a substance that
competes with glucose for binding to the transporter; for example, 6-Obenzyl-Dgalactose.Competition is not a feature of non-mediated transport, since no transport
protein is involved.
4.Inactivation-Reagents that chemically modify proteins and hence may affect their
functions may inhibit the rapid, saturatable flux of glucose into the erythrocyte.

Interesting facts:

The binding of the neurotransmitter acetylcholine at certain synapses opens

channels that admit Na+ and initiate a nerve impulse or muscle contraction.

Sound waves bending the cilia-like projections on the hair cells of the inner ear
open up ion channels leading to the creation of nerve impulses that the brain
interprets as sound.

Mechanical deformation of the cells of stretch receptors opens ion channels

leading to the creation of nerve impulses.

The crucial roles of the Na+/K+ ATPase are reflected in the fact that almost onethird of all the energy generated by the mitochondria in animal cells is used just to
run this pump.

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ABC transporters must have evolved early in the history of life. The ATP-binding
domains in archaea, eubacteria, and eukaryotes all share a homologous structure,
the ATP-binding "cassette".

Questions:
1. Carrier molecules that bring materials into cells are
a.

Lipids

b.

Proteins

c.

Glycogen

d.

Phospholipid

2. Arrange the following compounds in order of increasing membrane

permeability: N2, water, glucose and RNA.
a.

RNA>glucose>water>N2

b.

N2>water>glucose>RNA

c.

Water>N2>glucose>RNA

d.

N2>water>RNA>glucose

3. The rate of diffusion across the cell membrane is affected by the

a.

temperature and pinocytosis.

b.

temperature and size of the molecule.

c.

membrane structure and phagocytosis.

d.

shape of glycolipids and glycoproteins.

4. How many of the following factors would affect the permeability of the cell
membrane? Size of molecules Lipid solubility of molecules Presence of
transport channels Presence of ATP inside the cell.
a.

One.

b.

Two.

c.

Three.

d.

Four.

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5. Which of the following aids the movement of glucose across a cell membrane?
a.

Protein.

b.

Phosphate.

c. Glycolipid.
d. Cholesterol.
6. In the parietal cells of the stomach, the uptake of chloride ions is coupled to
the transport of bicarbonate ions out of the cell. This type of active transport
system is called,
a. Uniprot
b. Symprot
c. Antiprot
7. Which of the following conditions is required for diffusion to occur?
a. ATP energy.
b. A living cell.
c. A concentration difference.
d. A selectively-permeable membrane.
8. Frog eggs placed in an isotonic solution will
a. burst.
b. shrink.
c. remain the same.
d. increase in volume.
9. When put in a hypotonic environment, an animal cell will
a. swell.
b. shrink.
c. secrete enzymes.
d. remain unchanged.
10. Which of the following conditions would cause red blood cells to burst?
a. pH of 7.5.
b.

Temperature of 3C.

c. Being placed in distilled water.

d. Being placed in an 11% salt solution.

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11. In an experiment, frogs eggs were placed in a salt solution. After several
hours their mass increased significantly. We can therefore conclude that,
compared to the frogs eggs, the solution was
a. isotonic.
b. saturated.
c. hypotonic.
d. hypertonic.
12. Which of the following moves material against a concentration gradient?
a.

osmosis

b. diffusion
c. active transport
d. facilitated transport
13. Which of the following processes moves molecules using cellular energy?
a.

Osmosis.

b.

Diffusion.

c.

Pinocytosis.

d.

Facilitated transport.

14. Which of the following processes would be directly affected by a lack of

cellular ATP?
a. Osmosis.
b. Diffusion.
c. Active transport.
d. Facilitated transport.
15. Which of the following will be affected directly if the mitochondria in a cell
are not functioning properly?
a. Absorption of alcohol by the cell.
b. The movement of water into and out of the cell.
c. The movement of oxygen across the cell membrane.
d.

The movement of sugar from a low to a high concentration.

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16. The cell process which uses ATP to bring substances into the cell is
a.

Osmosis.

b.

Diffusion.

c. Active transport.
d.

Facilitated transport.

17. A bacterium is living in a pond where the concentration of sodium ions is

0.005mM. This ion is found in the bacterial cytoplasm at a concentration 0.10
mM. Therefore the sodium ion is probably entering by:
a. Simple diffusion
b. Facilitated diffusion
c. Passive transport
d. Active transport
18. What are the two factors that are responsible for diffusion rate?
19. What are the membrane potentials of living cells?
20. How the opening and closing of ion channels occur in a cell?
21. Explain glucose transporter or GLUT1 with a diagram.
22. What are the different types of mediated transport depending on the
thermodynamics of the system?
23. How the mediated transport can be differentiated from non-mediated
transport. Explain with a graph.
References:
1. Baldwin, S. A., J. C. Gorga, and G. E. Lienhard (1981); The monosaccharide
transporter of the human erythrocyte. Transport activity upon reconstitution, J.
Biol. Chem. 256:3685-3689.
2. Donald Voet, Judith G. Voet, Charlotte W. Pratt (2008); Fundamentals of
Biochemistry- Life at the Molecular Level: Chapter 10 Membrane transport, 3rd
Edition
3. Joyce

J.

Diwan

(1998-2007);

Membrane

transport,

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/carriers.htm
4. M. Lodish (2003), Molecular cell biology: Chapter 3 Biomembranes and cell
th

architecture, 5 edition
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5. M. Lodish (2003); Molecular cell biology: Chapter 7 Transport of ions and small
th

molecules across cell membranes, 5 edition

6. Sui H, han BG, Lee JK, Walian P, Jap Bk. (2001); Structural basis of waterspecific transport through the AQP1 water channel, Nature 414:872-878

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Module 3 Lecture 2
Membrane transport facilitators
Membrane transport is assisted by various facilitators to ease their job. We will study a
few of them in detail.
Permeases
Permeases are a class of membrane transport proteins which facilitate the diffusion of a
specific molecule by passive mediated transport. These are divided into following types:
1. Lactose permease: It is a transmembrane protein that consists of N- and C- terminal
domains, each consisting of six membrane-spanning alpha helices in a symmetrical
fashion. These two domains are well separated and are joined by a single stretch of
polypeptide. There are six side chains amino acids that play an important role in the
active transport of lactose through the protein. Some of the examples are: Glutamic Acid
126, Arginine 144, and Glutamic Acid 269 plays role in substrate binding activities where
as Arginine 302, Histidine 322, and Glutamic Acid 325 plays a significant role in proton
translocation throughout the transport process. These side chains, make up the active site
of the protein and found within the large internal hydrophilic cavity of the lactose
permease where the substrate is received for transport and it is the location from which it
is sent into the cell.
It is an active co-transport that facilitates the passage of lactose across the phospholipid
+

bi-layer of the cell membrane by using the inwardly directed H electrochemical gradient
as its driving force. The proton gradient is metabolically generated through oxidative
metabolism. The electrochemical potential gradient created by both these systems is used
mainly to drive the synthesis of ATP. As a result, the lactose is accompanied from the
+

periplasam to the cytoplasm of the cell by an H proton.

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Lactose permease has two major conformational states:

1. E-1, which has a low-affinity lactose-binding site facing the interior of the cell.
2. E-2, which has a high-affinity lactose-binding site facing the exterior of the cell.

Figure 1: Schematic diagram for the cotransport of H + and lactose by lactose permease in E.Coli. H+ binds first to E-2 outside
the cell, followed by lactose. They are sequentially released from E-1 inside the cell. E-2 must bind to lactose and H + in order to
change the conformation to E-1, thereby cotransporting these substances in the cell. E-1 changes the conformation to E-2 when
neither lactose nor H+ is bound, thus completing the transport cycle.

2. -galactoside permease is a membrane-bound transport protein that facilitates the

uptake of -galactosides across the cell. The common example is melibiose carrier
protein from Klebsiella pneumonia, which is capable of using hydrogen and lithium
cations as coupling cations for cotransport, depending on the particular sugar transported
+

(H -melibiose, Li -lactose).
3. Amino acid permeases are integral membrane proteins involved in the transport of
amino acids into the cell. One of the examples of amino acid permease is histidine
permease which is a bacterial ABC protein in E.coli and located in the periplasmic space
of cell. Histidine binding protein binds histidine tightly and directs it to T sub-units of
permease, through which hisitidine crosses the plasma membrane along with ATP
hydrolysis.

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+

Na /K ATPase :
+

In mammalian cells, the Na and K gradients are the two major components of the
electrochemical gradient across the plasma membrane. The cells maintain a lower
+

intracellular Na concentration and higher intracellular K concentration with relative to

extracellular space. Hence, for the generation and maintenance of the electrochemical
+

gradients for Na and K , it requires Na /K ATPase, which is an ion pump that couples
ATP hydrolysis to cation transport. It also helps to set the negative resting membrane
+

potential, which regulates the osmotic pressure to avoid cell lysis. The Na /K ATPase
belong to P-class ATPase which is commonly found in the plasma membranes of higher
eukaryotes. This transmembrane protein consists of two types of subunits: a 110-kD nonglycosylated - subunit that contains the enzymes catalytic activity and binding sites for
+

ATP, Na and K ions, and a 55-kD glycoprotein -subunit of unknown function. The
smaller -subunit has one transmembrane domain that stabilizes the -subunit and is
important in membrane insertion. The - subunit has eight transmembrane -helical
segments and two large cytoplasmic domains and the - subunit has a single
transmembrane helix and a large extracellular domain. The protein may function as an
()2 tetramer in vivo.

Figur 2: Na+/K+ ATPase. The diagram shows the transporters putative dimeric structure and its orientation in the plasma
membrane. Cardiotonic steroids bind to the external surface of the transporter, thereby inhibiting transport.

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+

The Na /K ATPase is also called as the Na /K pump because it pumps 3 Na out of and
+

2 K in both direction across the membrane in presence of hydrolysis of ATP. The overall
reaction is:
+

3 Na (in) + 2 K (out) + ATP + H2O 3 Na (out) + 2 K (in) + ADP + Pi

+

The important feature to the Na /K ATPase is the phosphorylation of a specific Asp

+

residue of the transport protein which phosphorylates only in the presence of Na ,

whereas the resulting aspartyl phosphate residue is subject to hydrolysis only in the
+

presence of K . Hence it has two conformations named E1 and E2. The protein appears to
operate in the following (explained in figure 4):
+

1. The protein in the E1 state has three high-affinities Na binding sites and two
+

low-affinity K binding sites accessible to the cytosolic surface of the protein.

+

Hence E1 binds three Na ions inside the cell and then binds ATP to yield an
+

E1 .ATP.3 Na complex.
2. ATP hydrolysis produces ADP and a high-energy aspartyl phosphate
+

intermediate E1-P.3 Na .
3. This high-energy intermediate relaxes to its low-energy conformation, E1~P.3
+

Na , and releases its bound Na outside the cell.

+

4. E2-P binds two K ions from outside the cell to form an E2-P.2 K complex.
+

5. The phosphate group is hydrolyzed, yielding E2 .2 K .

+

6. E2 .2 K changes conformation, releases its two K ions inside the cell, and
+

replaces them with three Na ions, thereby completing the transport cycle.

Figure 3: Scheme for the transport of Na+ and K+ by the Na+/ K+ ATPase.

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These are mostly target of a large number of toxins and important drug target. Some of
the examples are: the naturally occurring steroids called cardiac glycoside such as
+

ouabain and digitalis, inhibit ion transport by Na /K ATPase by binding reversibly to the
extracellular side of pump which in turn inhibit ATP hydrolysis and ion transport. Other
toxins like palytoxin from marine corals are also specific inhibitor. They block the
ATPase in an open state, allowing ions to flow down their concentration gradient, which
destroys electrochemical gradient.

Figure 4: Operational model of the Na +/K+ ATPase in the plasma membrane. Only one of the two catalytic subunits of this Pclass pump is depicted. It is not known whether just one or both subunits in a single ATPase molecule transport ions. Ion
pumping by the Na+/K+ ATPase involves phosphorylation, dephosphorylation, and conformational change. In this case,
hydrolysis of the E2P intermediate powers the E2 E1 conformational change and concomitant transpor t of two ions (K +)
inward. Na+ ions are indicated by red circles; K + ions, by purple squares; high-energy acyl phosphate bond, by ~P; low-energy
phosphoester bond, by P.

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Ca

2+

ATPase

Eukaryotic cells maintain a low concentration of free Ca

2+

in the cytosol (10

whereas the extracellular concentration is very high on the opposite face (10
Henceforth, a small influx of Ca

2+

-7

-3

M)
M).

significantly increases the concentration of free Ca

2+

2+

in the cytosol and the flow of Ca down its steep concentration gradient in response to
the extracellular signals is one of the means of transmitting these signals rapidly across
the plasma membrane. Hence cells maintain a steep Ca

2+

gradient across the plasma

membrane. The Ca2 ATPases are commonly found in muscle cells and neurons. The
skeletal muscle have specialized structure of large intracellular Ca
2+

sarcoendoplasmic recticulum which controls Ca

2+

stores called

uptake and release throughout the cell

2+

volume. These are mainly responsible for Ca extrusion from cytosol in muscle cells
which is required to stop muscle contraction and to initiate relaxation.
Ca

2+

transporters are the common example of P-type transport ATPase. It is also known

as Ca

2+

2+

pump or Ca

2+

ATPase or SERCA pump (Sacroendoplasmic recticulum Ca

2+

ATPase). These transporters actively pump Ca

the gradient. The structure of Ca

2+

out of the cell and helps in maintaining

pump has an asymmetrical arrangement of

2+

transmembrane and cytosolic domains that undergo movements during Ca

transport. It

contains 10 tranmembrane -helices and two cytoplasmic loops between the

2+

transmembrane -helices. The transmembrane -helices form Ca

binds two Ca

2+

binding site which

ions from cytosol. And the two cytoplasmic loops form three separate

domains: nucleotide binding domains that binds ATP, actuator domain that contains
catalytic phoshorylation site and P domain which is important for transmission of
conformational changes between cytosolic and transmembrane domains. In
unphosphorylated state, the two helices are disturbed and form a cavity for binding of
2+

two Ca

ions from the cytosolic side of the membrane. ATP also binds to a binding site

on the same side of the membrane and the subsequent transfer of the terminal phosphate
group of ATP to an aspartic acid of an adjacent domain lead to a drastic rearrangement of
the transmembrane helices. This rearrangement disturbs the Ca
releases Ca

2+

2+

binding site and

ions on the other side of the membrane that is into the lumen of SR. With

respect to figure 5 and 6, the mechanism of the Ca

2+

ATPase in the SR membrane can be

understood clearly through following steps:

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2+

1. The protein in E1 conformation has two high affinity binding sites for Ca

ions

accessible from the cytosolic side and ATP binds to a side on cytosolic surface.
2+

2. In the presence of Mg , the bound form of ATP is hydrolyzed to ADP and

phosphate. Later the liberated phosphate is transferred to a specific aspartate
residue in the protein, forming the high-energy acyl phosphate bond denoted by
E1 ~ P.
3. Then the protein undergoes a conformational change and generates E2, which has
two low-affinity Ca

2+

binding sites accessible to the SR lumen.

4. The free energy of E1 ~ P is greater than E2-P, and this reduction in free energy
leads to the E1 E2 conformational change. Simultaneously, the Ca

2+

ions also

dissociate from the low-affinity sites to enter the SR lumen, following which the
aspartyl-phosphate bond is hydrolyzed.
5.

Dephosphorylation then again leads to the E2 E1 conformational change, and

2+

E1 is ready to transport two more Ca

ions.

Figure 5: Scheme for the active transport of Ca2+ by the Ca 2+ ATPase. Here (in) refers to the cytosol and (out) refers to the
outside of the cell for plasma membrane Ca 2+ ATPase or the lumen of the endoplasmic reticulum (or sarcoplasmic reticulum)
for the Ca2+ ATPase of that membrane.

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Figure 6: Operational model of the Ca 2+ ATPase in the SR membrane of skeletal muscle cells. Only one of the two catalytic
subunits of this P-class pump is depicted. E1 and E2 are alternative conformations of the protein in which the Ca 2+ binding
sites are accessible to the cytosolic and exoplasmic faces, respectively. An ordered sequence of steps (1 6), as diagrammed
here, is essential for coupling ATP hydrolysis and the transport of Ca 2+ ions across the membrane. In the figure, ~P indicates a
high-energy acyl phosphate bond; P indicates a low-energy phosphoester bond.

Interesting facts:

The X-ray crystal structure of lactose permease was first solved in 2003 by J.
Abramson et al.

Ouabain is a cardiac glycoside toxin. Potent inhibitors that bind to potassium

+

binding sites. In the presence of Ouabain, Na /K ATPase cannot return to its

resting state.

One major type of gradient linked active permeases is the sodium-glucose

symport carrier.

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Questions:
1. Which of the following uses energy to transport molecules or ions against
their concentration gradient?
a. Voltage-gated Na+ channel
b. Acetylcholine receptor
c. Glucose transporter
d. ATP-ADP transporter
e. Na+/K+-ATPase
2. A membrane-spanning transporter protein that is also characterized as a
symporter would be involved in which one of the following transport
processes?
a.

Simple transport (e.g., lactose via Lac permease)

b. Simultaneous transport of one type of molecule into the cell and a different
molecule out of the cell (e.g., Na+ pump to move Na+ out of the cell)
c. Transport of potassium ions into the cell without any other ion or molecule being
transported in any direction
d. Unidirectional transport into the cell of only one type of molecule (found in very
low concentration in the periplasm) using the ATP-driven ABC translocation
system
3. The

sodium-potassium

pump

passes a. more Na+ out than K+ in

b. K+ out and Na+ in on a one-for-one basis
c. Na+ out and K+ in on a one-for-one basis
d. K+ and Na+ in the same direction
4. The sodium-potassium pump moves sodium and potassium ions against the
concentration gradient.
a. True
b. False

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+

5. The Na -K pump consumes a third of the total ATP supply of a typical

animal cell and is responsible for maintaining the high concentration of K

inside cells, for controlling cell volume, and for driving the uptake of sugars
and amino acids in the intestine and kidneys.
a. True
b. False
6. The energy needed to power the sodium-potassium pump is provided by the
a. Binding of ATP to the pump
b. Transport of ATP by the pump.
c. Splitting of ATP.
d. Formation of ATP.
7. Which of the following moves Ca

2+

back into the tubules of the SR after a

contraction?
+

a. The ATP-dependent H pump

b. The ATP-dependent myosin pump
c. Simple diffusion
+

d. The ATP-dependent Na /K pump

e. The ATP-dependent Na /K pump
f. The ATP-dependent calcium pump
8. SERCS

pumps

actively

transport

calcium: a. From ER to cytosol

b. From cytosol to ER
c. From extracellular space to the cytosol
d. From the cytosol to the extracellular space
e. From the mitochondria to the cytosol
+

9. In each cycle, the Na -K pump transfers ____K ions in the cell and ____
+

Na out of the cell.

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10. Bacterial lactose permease is a symporter of lactose and H+. When the
lactose concentrations in the cytosol and in the extracellular space are
identical but the pHs in the two locations are different as indicated below,
which direction would lactose be transported? Explain briefly why you think
that way.
Lactose permease

Cytosol
Extracellular
pH 5.0

pH 7.0

Cell membrane

11. Why the sodium-potassium transport mechanism is called a pump?

+

12. Explain Na /K pump with a schematic diagram.

2+

13. Explain Ca pump with a schematic diagram

14. What are the types of permeases?
References
1. Donald Voet, Judith G. Voet, Charlotte W. Pratt (2008); Fundamentals of
Biochemistry: Life at the Molecular Level: Chapter 10 Membrane transport, 3rd
Edition
2. M. Lodish (2003); Molecular cell biology: Chapter 7 Transport of ions and small
th

molecules across cell membranes, 5 edition

3. Toyoshima, C., M. Nakasako, H. Nomura, and H. Ogawa (2000); Crystal
structure of the calcium pump of sarcoplasmatic reticulum at 2.6 resolution,
Nature. 405:647655.

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Module 3

Lecture 3

Lysosome and vacuolar membrane

In earlier lecture we have studied about plasma membrane. However some cell organelles
have depending on the function which they perform have modified membranes. We will
study the membrane of a lysosome and vacuoles in detail in this lecture.
Lysosomes:
Lysosomes are central, acidic and membrane bound organelles that contain hydrolase
enzyme for the breakdown of all types of biological polymers- proteins, nucleic acids,
carbohydrates and lipids. They are mostly found in animal cells, while in yeast and
plants, it acts as lytic vacuoles. It is enclosed by membrane known as lysosomal
membrane that maintains the digestive enzyme at pH 4.5. Figure 1 shows the structure of
lysosome.

Figure 1: Lysosome

Functions of lysosomes:

Maintains pH by pumping protons from cytosol across the membrane via proton
pumps and chloride ion channels.

Protects the cytosol and rest of the cells from degradative enzymes within the
lysosome.

Acts as digestive system of the cell, serving both to degrade material taken up
from the outside of the cell and to digest obsolete components of cell itself.

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Sequestration of lysosomal enzymes.

Mediation of fusion events between lysosomes and other organelles.

Transport of degradation products to the cytoplasm

Lysosomal Membrane: To perform its function with efficacy the lysosomal membrane
needs some additional features in its membrane. It is slightly thicker than that of the
plasma membrane. It contains substantial amounts of carbohydrate component,
particularly sialic acid. In fact, most lysosomal membrane proteins are highly
glycosylated, which may help protect them from the lysosomal proteases in the lumen.
The lysosomal membrane has another unique property of fusing with other membranes of
the cell. This property of fusion has been attributed to the high proportion of membrane
lipids present in the micellar configuration. Surface active agents such as liposoluble
vitamins (A,K,D and E) and steroid sex hormones have a destabilizing influence, causing
release of lysosomal enzymes due to rupture of lysosomal membranes. Drugs like
cortisone, hydrocortisone and others tend to stabilize the lysosomal membrane and have
an anti-inflammatory effect on the tissue. The entire process of digestion is carried out
within the lysosome. Most lysosomal enzymes act in an acid medium. Acidification of
lysosomal contents depends on an ATP-dependent proton pump which is present in the
membrane of the lysosome and accumulates H+ inside the organelle. Lysosomal
membrane also contains transport proteins that allow the final products of digestion of
macromolecules to escape so that they can be either excreted or reutilized by the cell.
Lysosomal membrane composition:
+

The V-class H ATPase pump is generally present in lysosomal membrane. This class of
+

ATPase pump only transports H ions. Its main function is to acidify the lumen of the
organelles. The proton gradient between the lysosomal lumen (pH 4.55.0) and the
cytosol (pH 7.0) depends on ATP production by the cell.
These V-class proton pumps contain two domains: a cytosolic hydrophilic domain (V 1)
and a transmembrane domain (V0) with multiple subunits in each domain. Binding and
+

hydrolysis of ATP by the B subunits in V1 provides the energy for pumping of H ions

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through the proton-conducting channel formed by the c and a subunits in V0. These Vclass proton pumps are not phosphorylated and dephosphorylated during proton transport.
Figure 2 depicts a V-class proton pump.

Figure 2: V-class proton pump

These protons cannot acidify by themselves because a net movement of electric charge
+

occurs. Only a few protons build up positive H ions on exoplasmic face (inside) and for
+

each H pumped across, a negative ion will be left behind on cytosolic face, building
negative charged ions. These oppositely charged ions attract each other on opposite faces
of the membrane, generating a charge separation, or electric potential, across the
membrane. If more protons are pumped, the excess positive ions on exoplasmic face
+

repels other H ions and prevents pumping of extra proton long before a significant
+

transmembrane H concentration gradient had been established .

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(a)

(b)

Figure 3: Effect of proton pumping by V-class ion pumps on H+ concentration gradients and electric potential gradients across
cellular membranes. (a) If an intracellular organelle contains only V-class pumps, proton pumping generates an electric
potential across the membrane, luminal-side positive, but no significant change occurs in the intraluminal pH. (b) If the
organelle membrane also contains Cl - channels, anions passively follow the pumped protons, resulting in an accumulation of
H+ ions (low luminal pH) but no electric potential across the membrane.

Lysosomal membrane proteins:

Lysosomes are formed by the fusion of transport vesicles budded from Golgi network
with endosomes, which contain molecules taken up at the cell surface. And its membrane
proteins are usually highly glycosylated proteins decorating the luminal surface of
lysosomal membranes. They are most often known as lysosomal associated membrane
proteins (LAMP). LAMP-1, LAMP-2 and LIMP-2 are the most abundant components of
this membrane. And mainly involved in transport of newly synthesized hydrolases to the
lysosome (lysosomal integral membrane protein 2 (LIMP2)) and across the lysosomal
+

membrane (the V-type H -ATPase complex and chloride channel protein 7 (CLC7).

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Vacuolar membrane:
Vacuoles are the membrane bound sac within the cytoplasm which are filled with water
containing organic and inorganic molecules including enzymes and mostly present in
plants, fungi and some animals. This vacuole slowly develops as the cell matures by
fusion of smaller vacuoles (vesicles) derived from the endoplasmic reticulum and Golgi
apparatus.

Figure 4: Plant cell structure

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Figure 5: Animal cell structure

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Function of vacuoles:

Acts as storage organelles and contains water and small molecules. Stores salts,
minerals, nutrients, proteins, pigments, helps in plant growth, and plays an
important structural role for the plant.

Maintains internal hydrostatic pressure or turgor pressure within the cell

Maintains an acidic internal pH

Allows plants to support structures such as leaves and flowers due to the pressure
of the central vacuole. Also maintains turgor pressure against the cell wall.
Because of osmosis, water diffuses into the vacuole, and exerting pressure on the
cell wall. And water loss leads to shrinkage of the cell. Hence turgor pressure
needs to be maintained. Turgor pressure also dictates the rigidity of the cell and is
associated with the difference between the osmotic pressure inside and outside of
the cell.

In seeds, stored proteins needed for germination are kept in protein bodies, which
are modified vacuole.

Regulating the movements of ions around the cell.

Transports proton from cytosol to vacuole and hence stabilizes cytoplasmic pH

making the vacuolar interior most acidic by creating a proton motive force which
in turn used for the transport of nutrients into and out of the cell and allows
degradative enzymes to act.

Vacuoles also often store the pigments that give certain flowers their colors,
which aid them in the attraction of bees and other pollinators, but also can release
molecules that are poisonous, odoriferous, or unpalatable to various insects and
animals, thus discouraging them from consuming the plant.

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Plant vacuoles:
Most of the plant cell contains large, single central vacuoles and can occupy at least 30%
to 80% of the cell. Generally vacuole is surrounded by membrane known as tonoplast, or
vacuolar membrane. It separates the vacuolar contents from cells cytoplasm and an
important and highly integrated component of the plant internal membrane network
(endomembrane) system. The vacuole solution (also known as cell sap) differs markedly
from that of the surrounding cytoplasm.
Vacuolar membrane:
The V-class H+ ATPase pump is present in vacuolar membrane. More details of V-class
H+ ATPase pump is described earlier (Figure 2 and Figure 3).

Figure 6: Plant cell vacuole

Other transport proteins present in vacuolar membrane:

1. Proton Pump:
Proton pumps play a central role in the function of the tonoplast by generating a
transmembrane H+ electrochemical gradient which can be utilized to drive the transport
of solutes. The tonoplast contains different proton pumps, an ATPase and a PPase. VATPases (vacuolar-type) are present on different membranes of eukaryotic cells and is
constituted of 13 subunits whereas tonoplast PPase is also an integral entity of the
tonoplast and consists of one 80 kDa protein.

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2. Aquaporins:
Major intrinsic membrane proteins (MIPs), are very small hydrophobic proteins
abundantly present in membranes. But these MIPs form water channels. Later -TIP
(tonoplast intrinsic protein) which is a member of MIPs was described and found
abundantly. Another major membrane protein of the central vacuole is the -TIP
(observed in radish). Both TIPs have been shown to act as water channels. -TIP is
associated with the storage vacuole while the -TIP is localized on the lytic vacuole.
Interestingly, -TIP has to be phosphorylated in order to exhibit water channel activity.
3. ABC transpoters:
Another class of transporters are ABC type transporters, which are directly energized by
MgATP and do not depend on the electrochemical force. Their substrates are organic
anions formed by conjugation, e.g. to glutathione.
Examples of solute transport across vacuolar membrane in plant cells:
Transport of products of primary metabolites:
The various types of Primary metabolites could be:
1. Carbohydrates: Sucrose uptake occurs by facilitated diffusion in leaf vacuoles. Later
it was also observed that active transport of sucrose takes place for vacuoles isolated from
sugar cane cell cultures, which accumulates sucrose at concentrations comparable to
those in the stalk tissue and tomato fruit vacuoles. Furthermore, it was also found that
sucrose transport was stimulated by MgATP and to occur via a sucrose/H+ antiport in red
beet. Larger carbohydrates such as stachyose, which is present in large quantities in
Stachys sieboldi, may also be accumulated in the vacuole by proton antiport mechanisms.
Many sugar alcohols also found in plants accumulate within the vacuoles. Transport of
sorbitol across the tonoplast appears to be ATP-dependent in case of immature apple fruit
tissue. Transport experiments suggest that mannitol crosses the tonoplast by facilitated
diffusion.
2. Amino acid:
The first amino acid transport system was observed in barley plants. These are carriers or
channles which are modulated by free ATP (but not by MgATP) which induces inward as
well as outward fluxes of all amino acids tested.

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3. Organic acids:
With context to organic acids, malate transport across the vacuolar membrane has been
studied most intensively. This is due to the central role of malate in plant metabolism.
The uptake of maltate is mainly governed by the electrical component of the
electrochemical potential generated by the proton pumps. This channel also mediates
uptake of succinate, fumarate, and oxaloacetate. The malate channel is not affected by
cytosolic Ca2+ or ATP and it is a 32 kDa subunit protein. Citrate crosses the tonoplast
using the same transporter as malate.
4. Inorganic anions:
The H+ pumps generate a positive potential inside the vacuole, which is the driving force
for anion movements. Anion-dependent dissipation of a proton-pump generated by anions

2-

revealed that NO3 permeates more rapidly than Cl and SO4 whereas HPO4 crossed
the tonoplast considerably slowly.
Chloride:

An ATP-dependent Cl uptake was studied in barley mesophyll vacuoles. Later a

vacuolar Cl channel (VCL) was identified in Vicia faba guard cells which is activated by
+

a calcium dependent protein kinase (CDPK) in the presence of ATP and Ca2 and, to a
weaker extend (22%), by protein kinase A. The VCL channel was activated at

physiological potentials enabling Cl uptake into vacuoles.

Nitrate:
Amongst the anions it exhibits the highest permeability through the vacuolar membrane.
It was concluded in one of the experiment that a membrane potential driven nitrate

transporter, a NO3 /H antiporter is present on the tonoplast.

Sulphate:
Using tonoplast vesicles, it has been shown that SO4
tonoplast slowly as compared to NO 3

and HPO4

anions cross the

or Cl . It has been found that SO 4

uptake is

stimulated by Mg -ATP.

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Phosphate:
Pi starvation leads to an efflux of Pi from the vacuole. It has been shown that Pi
concentrations in the cytosol are maintained at a constant level in Acer pseudoplatanus
cells using

31

P NMR.

5. Inorganic cation:
The membrane potential of the cytosol with respect to the vacuole is negative (2040
mV). This implies that cations are excluded from the vacuole unless transport is coupled
to an energy-dependent uptake mechanism.
Potassium:
Several channels exhibiting potassium permeability have been described. The first
channel demonstrated for vacuolar membrane was called SV (slow activating vacuolar)
channel. This channel is a slow activated channel and is associated with Ca
+

calmodulin-induced K and Ca
+

permeability of Na if Ca

2+

2+

2+

and

fluxes. These channels have been reported for the

concentrations are increased by a signal. Secondly, FV (fast

vacuolar) channel activates instantaneously in response to voltage changes. These

+

channels may allow the release of K at low Ca

2+

concentrations. Thirdly, the vacuolar

K (VK) channel is activated instantaneously but it can be distinguished from the FV

channel. It is voltage independent and fully activated at low cytosolic pH.
+

A K /H antiport mechanism has been also reported for tonoplast enriched fractions from
zucchini, Brassica napus hypocotyls, and Atriplex.
Sodium:
+

Na accumulation is accompanied by vacuolar alkalinization in barley roots. This was

established by using NMR spectroscopy.
Calcium:
Calcium plays a central role in signal transduction and higher concentrations are observed
in apoplast and within the vacuole. An energized, highly specific calcium uptake by the
vacuole is, therefore, a prerequisite for maintaining a low cytosolic calcium
concentration. P-type Ca-ATPases have been identified at the plasma membrane, the ER,
and the vacuolar membrane. A Ca

2+

2+

pump called a Ca /H

antiporter has been

demonstrated in vacuolar membrane fractions. This antiporter exhibits a far lower affinity

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2+

than the Ca -ATPase. Also, a vacuolar voltage gated Ca

2+

channel (VVCa) has been

reported which is activated on membrane hyperpolarization.

Magnesium:
2+

The presence of a Mg /H antiporter has been described for the vacuole-like lutoids of
Hevea brasiliensis and tonoplast vesicles isolated from maize roots.
Heavy metals:
Plants need some heavy metals such as Cu

2+

or Zn

2+

as micronutrients. Therefore, they

need to be transported and a large portion of the heavy metals absorbed by the cell is
2+

usually concentrated within the vacuole. A vacuolar Cd /H antiport activity has been
demonstrated. However, it is known that plants form chelates with heavy metals by
synthesizing phytochelatins (PCs), and these PCs can be transported into vacuoles of
Schizosaccharomyces as apoPC or as PC-Cd complexes by ABC transporters. Vacuoles of
higher plants are also known to transport phytochelatins.
Transport of products of the secondary metabolites: Involvement of secondary
energized transporters and directly energized, ABC-type transporters
Plants synthesize an huge number of secondary metabolites and many of these have been
found to be exclusively localized in the vacuole. The electrochemical gradient established
by the two vacuolar proton pumps is used by the secondary energized transporters as a
source of energy. It was demonstrated that the pH was essential for the uptake of a
number of phenolics, such as esculin, o-coumaric acid glucoside, apigenin- 7-(6-Omalonyl) glucoside, and anthocyanins from carrot. Recently it became evident that in
addition to transporters depending on the proton motive force, directly energized
transporters are also present on the vacuolar membrane. The first demonstration for a
directly activated transport of solutes into the vacuole was provided for glutathione
conjugates. Flavonoid glucuronides, a secondary plant compounds in rye vacuoles are
transported by directly energized transport processes. Furthermore, studies with lucifer
yellow, a sulfonated compound also indicates that sulfonated and sulfated secondary
compounds cross the tonoplast by direct energization.

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Figure 7: Proton pumps establishing a electrochemical gradient (red), secondary energized uptake mechanisms (green), and
directly energized, ABC-type transporters (blue) of the plant vacuole. S, neutral solute; A, anion; cat+, cation; X-conjugate,
conjugate of a compound X (secondary metabolite or xenobiotic) with a hydrophilic compound such as glucose, glutathione, an
amino acid, malonate, or sulphate.

Interesting facts:

During endocytosis, these intra-lysosomal membranes are formed and prepared

for digestion by a lipid-sorting process during which their cholesterol content
decreases

and

the

concentration

of

the

negatively

charged

bis(monoacylglycero)phosphate increases.

Lysosomal enzyme disorders contribute to several human diseases, either due to

genetic defects in its enzyme expression or the escape of lysosomal enzymes
(lysozymes) into extralysosomal medium.

Permeabilization of lysosome, has been shown to initiate a cell death pathway or

apoptosis.

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Questions
1. Which pump is present in lysosomal membrane?
a. P-class pump
b. ABC transporter
c. V-class pump
d. F-class pump
2. The pH of the lysosomal compartment is
a. 4
b. 4.6
c. 5
d. 5.6
3. Which of the following correctly matches an organelle with its function?
a. mitochondrion.photosynthesis
b. Nucleus.cellular respiration
c. Ribosome.manufacture of lipids
d. Lysosome.movement
e. Central vacuole.storage
4. Lysosomes are reservoirs of
a. Hydrolytic enzymes
b. Fat
c. Secretory glycoproteins
d. RNA
5. A function of lysosomes is
a. Syntheisis
b. Hydrolysis
c. Replication
d. Respiration

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6. For digestion to occur in a vacuole, the vacuole must first fuse with
a. Nucleus
b. Ribosome
c. Lysosome
d. Golgi bodies
7. Lysosomes can be expected to be present in large numbers in cells which
a.

Have cilia.

b.

Produce centrioles.

c.

Are actively dividing.

d.

Carry out phagocytosis.

8. For digestion to occur in a vacuole, the vacuole must first fuse with
a. Nucleus
b. Ribosome
c. Lysosome
d. Golgi body
9. The proton gradient between the lysosomal lumen (pH 4.55.0) and the cytosol
(pH 7.0) depends on ATP production by the cell.
a. True
b. False
10. What is the function of permanent vacuole?
a. Supports and protects the cell
b. Controls what enters and leaves the cell
c. Controls the cell
d. Stores water and mineral ions
e. Stores water and mineral ions
11. Vacuole is surrounded by membrane called
a. Tonoplast
b. Chloroplast
c. Plasma membrane
18. What are the most abundant components of lysosomal membrane?
19. Write the composition and functions of vacuolar membrane.

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References:
1. Enrico Martinoia, Agns Massonneau and Nathalie Frangne (2000); Transport
Processes of Solutes across the Vacuolar Membrane of Higher Plants, Plant Cell
Physiol 41 (11): 1175-1186
2. M. Lodish (2003); Molecular cell biology: Chapter 3 Biomembranes and cell
th

architecture, 5 edition
3. M. Lodish (2007); Molecular cell biology: Chapter 7 Transport of ions and small
th

molecules across cell membranes, 5 edition

4. Paul Saftig (2005); Lysosomes: Lysosomal membrane proteins

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Module 3 Lecture 4
ATP dependent proton pumps
Proton pump
The proton pump is a transmembrane protein that is capable of transport of protons across
the cell membrane, mitochondria and other cell organelle.
ATP dependent proton pumps
+

ATP dependent proton pumps or transport ATPase are the pumps that transport H ions
against their concentration gradients. These pumps are transmembrane proteins with one
or more binding sites for ATP located on the cytosolic face of the membrane and these
proteins are called ATPases. They normally do not hydrolyze ATP into ADP and Pi unless
+

H ions are simultaneously transported. Because of this tight coupling between ATP
hydrolysis and transport, the energy stored in the phosphoanhydride bond is not
dissipated but rather used to move ions or other molecules uphill against an
electrochemical gradient.
ATP dependent proton pumps can be categorized into different classes. Generally, ATP
dependent proton pumps are divided into 4 classes:

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Figure 1: Different types of ATP dependent proton pumps

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1. P-class ion pumps:

These are multipass transmembrane proteins having two identical catalytic -subunits
that contain an ATP binding site. Some have two smaller -subunits that usually have
regulatory functions. During the transport process or pumping cycle at least one of the +

subunit must be phosphorylated and the H ions are thought to move through the
phosphorylated subunit. This class includes many ion pumps that are responsible for
+

setting up and maintaining gradients of Na , K , H and Ca2 across the cell membrane.
a) The common P-type pump is mostly found in parietal cells of the mammalian stomach
+

which transport protons (H ions) out of cell and K ions into the cell and is mainly
+

responsible for the acidification of the stomach contents. The pump is known as H /K
+

ATPase. It is a heterodimeric protein. The H /K ATPase transports one H+ from the

+

cytoplasm of the parietal cell in exchange for one K retrieved from the gastric lumen. As
+

an ion pump the H /K ATPase is able to transport ions against a concentration gradient
using energy derived from the hydrolysis of ATP. Like all P-type ATPases, a phosphate
+

group is transferred from ATP to the H /K ATPase during the transport cycle.
+

b) Another example of P-type pump is Na /K ATPase in the plasma membrane, which

+

generates low cytosolic Na and high cytosolic K concentration which is typical of

animal cells (discussed in earlier lecture).
c) Certain Ca

2+

ATPase pump Ca

2+

ions out of the cytosol into the external medium while

others pump Ca2+ from the cytosol into the endoplasmic recticulum or into the
specialized sarcoplasmic reticulum, which is more common in muscle cells (discussed in
earlier lecture).
2. F-class ion pumps:
The F class ion pumps contain different transmembrane and cytosolic subunits. They are
known for only transport of protons, in a process that does not involve phosphoprotein
intermediate. They generally behave as reverse proton pump by synthesizing ATP from
ADP and Pi by movement of protons from the exoplasmic to the cytosolic face of the
membrane down the proton electrochemical gradient. Therefore, these pumps are also
known as ATP synthases or F0F1 complex. F-class ion pump is most common in bacteria,
yeast and animal mitochondria and also in chloroplast.

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The F0F1 complex is a multi-protein having two components F 0 and F1. Both are
multimeric proteins. The F0 component contains three integral membrane proteins named
a, b and c. The a and two b subunits are linked tightly but not to the donut- shaped ring of
c subunits. And the F1 component is water soluble complex of five distinct polypeptides
with the composition 33. The lower part of the F 1 subunit is a coil which fits into
the centre of the c-subunit ring of F 0 and appears rigidly attached to it. The F1 subunit is
rigidly attached to and also forms rigid contacts with c subunits. The F 1 and subunits
associate in alternating order to form a hexamer . The F 1 subunit is permanently
linked to one of the F1 subunits and also to the b subunit of F 0. Thus the F0 a and b
subunits and the subunit and ()3 hexamer of the F 1 complex form a rigid structure
anchored in the membrane. The rodlike b subunits form a stator that prevents the ()3
hexamer from moving while it rests on the subunit.

Figure 2: Model of the structure and function of ATP synthase (the F0F1 complex) in the bacterial plasma membrane.

The F0 portion is built of three integral membrane proteins: one copy of a, two copies of
b, and on average 10 copies of c arranged in a ring in the plane of the membrane. Two
proton half-channels lie at the interface between the a subunit and the c ring. Halfchannel I allows protons to move one at a time from the exoplasmic medium and bind to
aspartate-61 in the center of a c subunit near the middle of the membrane. Half-channel II
(after rotation of the c ring) permits protons to dissociate from the aspartate and move
into the cytosolic medium.

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3. V-class ion pumps:

It is almost similar to F-class ion pumps in structure and function. But none of their
subunits are related to each other. F-class pumps operate in reverse direction to F-class.
These pumps generally function to maintain low pH of plant vacuoles and lysosome and
other acidic vesicles in animal cells by pumping protons from cytosolic to exoplasmic
face (inside) of membrane against the proton electrochemical gradient. The acidification
between the lysosomal lumen and cytosol lumen can be maintained by production of ATP
by cells.
These V-class proton pumps contain two domains: a cytosolic hydrophilic domain (V 1)
and a transmembrane domain (V0) with multiple subunits in each domain. Binding and
+

hydrolysis of ATP by the B subunits in V 1 provide the energy for pumping of H ions
through the proton-conducting channel formed by the c and a subunits in V0. These Vclass proton pumps are not phosphorylated and dephosphorylated during proton transport.

Figure 3: V-class proton pump

These protons cannot acidify by themselves because a net movement of electric charge
+

occurs. Only a few protons build up positive H ions on exoplasmic face (inside) and for
+

each H pumped across, a negative ion will be left behind on cytosolic face, building
negatively charged ions. These oppositely charged ions attract each other on opposite
faces of the membrane, generating a charge separation, or electric potential, across the
membrane. If more protons pumped, the excess positive ions on exoplasmic face repels
other H

ions and prevents pumping of extra proton long before a significant

+

transmembrane H concentration gradient had been established. If the organelle lumen or

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the extracellular space has to be acidified, the net movements of protons must be
-

accompanied either by movement of equal number of anion eg Cl in same direction or my

movement of different cation in the opposite direction. The first process occurs in lysosomes
+

and plant vacuoles whose membrane contains V-class H ATPase and anion channels for Cl

movement. And the second process is observed in the lining of the stomach which contains a
+

H /K ATPase and pumps one H outward and one K inward.

Figure 4: Effect of proton pumping by V-class ion pumps on H+ concentration gradients and electric potential gradients across
cellular membranes. (a) If an intracellular organelle contains only V-class pumps, proton pumping generates an electric
potential across the membrane, luminal-side positive, but no significant change in the intraluminal pH. (b) If the organelle
membrane also contains Cl- channels, anions passively follow the pumped protons, resulting in an accumulation of H + ions
(low luminal pH) but no electric potential across the membrane.

4. ABC (ATP binding cassettes) superfamily:

The final class of ATP-powered pumps is a large family of multiple membranes. This
class includes several hundred different transport proteins found in all organisms ranging
from bacteria to mammals. Each ABC protein is specific for single substrate or group of
related substrate, which may be ions, sugars, amino acids, phospholipids, cholesterol,
peptides, polysaccharides or proteins. All ABC transport protein share a structural
organization consisting of four core domains: two transmembarne (T) domains, forming

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the passageway through which transported molecules cross the membrane and two
cytosolic ATP-binding (A) domains. The core domains are generally present in separate
polypeptides which are more common in bacterial cell. In others, the core domains are
fused into one or two multidomain polypeptides. ATP binding leads to dimerization of
two ATP-binding domains and ATP hydrolysis leads to their dissociation. Theses
structural changes in the cytosolic domains are thought to be transmitted to the
transmembrane segments, driving cycles of conformational changes that alternately
expose substrate-binding sites on one side of the membrane and then on the other. In this
way, ABC transporters use ABC binding and hydrolysis to transport small molecules
across the bilayer. Some common example of ABC transporters are found in bacterial
plasma membranes which contain amino acid, sugar and peptide transporters. These cells
+

use H gradient across the membrane to pump variety of nutrients into the cell. It is also
present is mammalian plasma membrane that contains transporters of phospholipids,
small lipophillic drugs, cholesterol and other small molecules. One example of eukaryotic
ABC transporters is multidrug resistance (MDR) protein which has the ability to pump
hydrophobic drugs out of the cytosol. Overexpression of these MDR protein in human
cancer cells, make the cells resistant to variety of chemically unrelated cytototoxic drugs.
Interesting facts:

Valinomycin is a carrier for potassium.

Lactose permease has been crystallized with thiodigalactoside (TDG), an analog

of lactose.

Adenine nucleotide translocase (ADP/ATP exchanger), which catalyzes 1:1

exchange of ADP for ATP across the inner mitochondrial membrane.

The reaction mechanism for a P-class ion pump involves transient covalent
modification of the enzyme.

Gramicidin is an example of a channel. It is an unusual peptide, with alternating D

and L amino acids. In lipid bilayer membranes, gramicidin dimerizes and folds as
a right handed -helix. The dimer just spans the bilayer.

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Questions
1. The functional mechanism of P-class ion pumps is ............... by the ATP.
2. V-class pumps pumps exclusively ...................
3. Substance concentration + electric potential = ............................. which
determines the energetically favorable direction of transport a charged molecule
across a membrane.
4. Differentiate among Transporters, pumps and channels.
5. Is calcium pump and ATP dependent proton pump are same?
6. Describe ABC (ATP binding cassettes) superfamily.
7. Differentiate between V class proton pump and P-class ion pumps.
8. What are F-class ion pumps? How do they differ from the other classes of ion
pumps?
9. What is the main function of a V-class proton pump?
10. Give atleast three examples of ATP-binding cassettes.
11. Give a brief overview of the structural organization of the ABC transport proteins.
References:
1. M. J. Schnitzer (2001); Molecular motor: Doing a rotary two-step; Nature
410:878, and P. D. Boyer, 1999, Nature 402:247.
2. M. Lodish (2003); Molecular cell biology: Chapter 7 Transport of ions and small
th

molecules across cell membranes, 5 edition

3. M. Lodish (2003); Molecular cell biology: Chapter 8 Cellular energetic, 5
edition

th

4. Nishi, T., and M. Forgac. (2002); The vacuolar (H )-ATPases natures most
versatile proton pumps; Nature. Rev. Mol. Cell Biol.
5. Toyoshima, C., M. Nakasako, H. Nomura, and H. Ogawa (2000); Crystal
structure of the calcium pump of sarcoplasmic reticulum at 2.6 resolution;
Nature 405:647655.

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Module 3 Lecture 5
Cotransport: Symport, Antiport
Transporters:
Transporters (also known as carriers) are the membrane proteins that transport a wide
variety of ions and molecules across the lipid bilayer membrane.
Cotransporters:
Cotransporters are proteins that transport two different solutes such as glucose and amino
acids simultaneously across the cell membrane against a concentration gradient. It
mediates coupled reactions in which an energetically unfavorable reaction (uphill
movement of molecules) is coupled to an energetically favorable reaction. Unlike ATPase
pump, it uses the energy stored in electrochemical gradient. This is called secondary
mediated active transport (discussed in earlier lecture). An important feature is that
neither molecule can move alone; movement of both molecules together is obligatory, or
+

coupled. One of the common example is the energetically movement of Na ions into the
cell across the plasma membrane driven both by its concentration gradient and by the
transmembrane voltage gradient, which can be coupled to movement of the transported
molecule (glucose) against its concentration gradient.
How cotransportersare differentiated from uniporters?
Both transporters share some common feature with respect tostructural similarities,
operation at equivalent rates, and undergo cyclical conformational changes during
transport of their substrates. They differ in that uniporters can only accelerate
thermodynamically favourable transport down a concentration gradient, whereas
cotransporters can harness the energy of a coupled favourable reaction to actively
transport molecules against a concentration gradient.
Types of cotransports:
On the basis of movement of solutes, cotranporters can be divided into following
categories:
1. Symport:When the transported molecule and cotransported ion move in the same
direction, the process is called symport.
2. Antiport:When the transported molecule and cotransported ion move in the
opposite direction, the process is called antiport.

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Both the above mentioned cotranporter move one solute against its transmembrane
concentration gradient. This movement is powered by coupling to the movement of
second solute down its transmembrane concentration gradient.

Figure 1: Transporters, which fall into three groups, facilitate movement of specific small molecules or ions.(A) Uniporters
transport a single type of molecule down its concentration gradient. Cotransport proteins (symporters (B), and antiporters
(C)) catalyze the movement of one molecule againstits concentration gradient (black circles), driven by movement of one or
more ions down an electrochemical gradient (red circles). Differences in the mechanisms of transport by these three major
classes of proteins account for their varying rates of solute movement.

On the basis of movement of ions, cotransporters can also be categorized into:

+

1. Cationcotransporter:Example of cation transporter is Na /H antiporter, which

+

exports H from cells coupled to the energetically favorable import of Na .

-

2. Anion cotransporter:Example of anion transporter is exchange of Cl and HCO3

across the plasma membrane.

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Some common examples of cotranporter are:

+
1. Oligosaccharide/H symporter:They are also known as LacYsymporter. It is most
common in bacterial cell which has lactose permeaseLacY and functions as
symporter. It uses the free energy released from translocation of H

down its

electrochemical gradient to drive the accumulation of nutrients such as lactose against

+

its concentration gradient. The H gradient across the cytoplasmic membrane is

established by the respiratory chain and by the action of F 1F0-ATPase, which couples
ATP hydrolysis to the export of protons from the cell. For LacY, the stoichiometry of
+

lactose and H translocation is 1:1, with both substances movement in the same
direction. Thus, the lactose gradient can drive the uphill translocation of protons and
+

generate an inward or outward H gradient, depending on the direction of the lactose

concentration gradient.
Structure of Lac Y symporter:Structurally theLacYsymporter contains 12
transmembarne helices which are connected by hydrophilic loops and cytoplasmic Nand C- termini. There are two domains of 6 transmembranesegments each, forming a
symmetrical structure. The hydrophilic cavity which lies in the centre of lipid bilayer
forms the substrate binding site. This substrate binding site is accessible from either
the intracellular or extracellular side of the membrane but never to both sides
simultaneously. Protonation and binding of lactose in the outward-facing
conformation

induces

conformation

change,

resulting

in

inward-facing

conformation. This structural arrangement involves binding of both substrates

initially and allows for coupled and then simultaneous transport. Release of lactose
and protons into the cell then induces a transition back to the outward-facing
conformation. Hence it lowers the energy barrier between inward and outward-facing
conformation and facilitates interconversion.
2. Glycerol-3-phosphate transport (GlpT):It is an antiporter that accumulates
glycerol-3-phosphate into the cell for energy production and phospholipid synthesis.
GlpT is an organic phosphate/inorganic phosphate exchange which is driven by Pi
gradient. Similar to LacY, it has also symmetrical N- and C- terminal domains, each
consisting of 6 transmembrane segments surrounding the substrate translocation
pathway. It also works as same mechanism asLacY but glycerol-3-phosphate binds

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and phosphate is released in the outward conformation and opposite occurs in the
inward conformation.
+

3. Na linked symporter:This symporter imports amino acid and glucose into the
animal cells against the concentration gradient. Anexample is GLUT protein which
imports glucose from the blood downs its concentration gradient. On the other hand,
certain cells such as those lining the small intestine and kidney tubules, import
glucose from intestinal lumen or forming urine against a large concentration gradient.
+

Such cells utilize two Na /one glucose symporter, a protein that couples to import one
+

glucose to import two Na . This symporter contains 14 transmembrane helices with

both its N- and C- termini extending in the cytosol. The N-terminal portion of the
+

protein, including helices 19, is required to couple Na binding and influx to the
transport of glucose against a concentration gradient.
+

The following steps occur for transport of Na and glucose:

+

1. Simultaneous binding of Na and glucose to the conformation with outwardfacing binding sites
2. A second conformation generates with inward facing side
+

3. Dissociation of Na and glucose into the cytosol

4. The protein reverts back to original outward-facing conformation, ready to
transport the next substrate

Figure 2: Operational model for the two-Na+/one glucose symporter.

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The overall reaction is:

+

2Na (out) + Glucose (out) 2Na (in) + Glucose (in)

+

The free energy change for the symport transport of two Na and one glucose is the sum
of the free energy changes generated by the glucose concentration gradient (1 molecule
+

transported), the Na concentration gradient (2 Na ions transported), and the membrane

+

potential(generated by two Na tranported):

[

] +2

] +2

When G=0 and the free energy released by movement of Na

into cells down its

electrochemical gradient has a free energychange G of about -3 kcal per mole of Na

transported.Thus the G for transport of two moles of Na inward is about -6 kcal. By

substituting in above equation, the ratio of glucose (in)/glucose (out) =30,000. Thus if 2
+

moles of Na inward then it generates an intracellular concentration of glucose of 30,000

+

times more than extracellular glucose. Thus if only one Na ion were imported per glucose
molecule, then the available energy could generate a glucose concentration gradient (inside+

outside) of only about 170-fold. Thus by coupling the transport of two Na ions to the
+

transport of one glucose, the two-Na /one-glucose symporter permits cells to accumulate a
very high concentration of glucose relative to the external concentration.
+

4. Na linked antiporter:A cotranporter, 3Na /Ca2 antiporter in cardiac muscle cell

maintain a low concentration of Ca
transporter is:
+

2+

2+

in cytosol. The reaction for this cation

2+

3Na (out) + Ca (in) 3Na (in) + Ca (out)

+

The movement of three Na ions is required to power the export of one Ca

2+

-7

2+

ion from the

2+

cytosol with a [Ca ] of 2 * 10 M to the extracellular medium with a [Ca ] of 2 * 10

2+

-3

M, a gradient of some 10,000-fold form. By lowering cytosolic Ca , operation of the

+

2+

Na /Ca antiporter reduces the strength of heart muscle contraction.

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Function of cotransporter:
1. Regulation of cytosolic pH:
The anaerobic metabolism of glucose yields lactic acid whereas the aerobic metabolism
yields CO2, which reacts with water to form carbonic acid (H2CO3). This weak acid
+

dissociates yielding H ion or proton. If these excess protons werenot removed from
cells, then the cytosolic pH would drop and will be unfavourable to cellular fractions.
+

Hencecotransports are required to remove excess of protons. One is Na HCO3 /Cl

+

antiport imports one Na down its concentration gradient together with one HCO3 in
-

exchange for export of one Cl against its concentration gradient. The enzyme named
-

carbonic anhydrase catalyzes dissociation of imported HCO 3 ions into CO2 and OH by
the reaction:
3-

HCO CO2 + OH
-

OH + H H2O
-

Then CO2 diffuses out of the cell and OH ions combine with intracellular protons,
+

forming water. Thus the overall action of this transport is to consume cytosolic H ions,
thereby raising cytosolic pH.
+

Secondly Na /H antiporter plays an important role in raising cytosolic pH which couples

+

entry of one Na into the cell down its concentration gradient to export of one H ion.
-

Thirdly, anion antiporterthatcatalyzes the one-for-one exchange of HCO3 and Cl across

-

the plasma membrane. At high pH, this Cl /HCO3 antiporterexports HCO3 in exchange
-

for Cl , thus lowering the cytosolic pH. The import of Cl down its concentration gradient
-

(Cl (medium) >Cl (cytosol)) powers the reaction.

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The activity of all these antiports depends upon pH.The two antiportersthat operate to
increase cytosolic pH are activated when the pH of the cytosol falls. Similarly, a rise in
-

pH above 7.2 stimulates the Cl /HCO3 antiporter, leading to a more rapid export of
-

HCO3 and decrease in the cytosolic pH. In this manner the cytosolic pH of growing cells
is maintained very close to pH 7.4.

Figure 3: The activity of membrane transport proteins that regulate the cytosolic pH of mammalian cells changes with
pH.Direction of ion transport is indicated above the curve for each protein

2. Accumulation of metabolites and ions inplant vacuoles:

The lumen of plant vacuoles is much more acidic(pH 3 to 6) than is the cytosol (pH 7.5).
-

The vacuolarmembrane contains Cl and NO3 channels that transportthese anions from
the cytosol into the vacuole against their concentration gradients and is drivenby the
+

inside-positive potential generated by the H pumps.

One more example is proton/sucrose antiporterin the vacuolar membrane that
accumulate sucrose in plant vacuoles. During photosynthesis, sucrose is generated and
stored in vacuole. But during night these stored sucrose moves into the cytoplasm and is
metabolized to CO2 and H2O with generation of ATP from ADP and Pi. The inward
movement of sucrose is governed by movement of H

which is favoured by its

concentration gradient(lumen cytosol) andby the cytosolic-negative potential across the

vacuolar membrane.

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Uptake of Ca

2+

and Na into the vacuolefrom the cytosol against their concentration

gradients issimilarly mediated by proton antiporters.

Figure 4: Accumulation of ions and sucrose by the plant vacuole.The vacuolar membrane contains two types of proton pumps
(orange): a V-class H+ ATPase (left) and a pyrophosphate-hydrolyzing proton pump (right ) that differs from all other iontransport proteins and probably is unique to plants. These pumps generate a low luminal pH as well as an inside positive
electric potential across the vacuolar membrane owing to the inward pumping of H + ions. The inside-positive potential powers
the movement of Cl- and NO3- from the cytosol through separate channel proteins (purple). Proton antiporters (green),
powered by the H+ gradient, accumulate Na+, Ca2+, and sucroseinside the vacuole.

Interesting facts:

Na+/K+/2Cl- symporter in the loop of Henle in the renal tubules of the kidney
transports 4 molecules of 3 different types; a sodium ion (Na+), a potassium ion
(K+) and two chloride ions (2Cl-).

In the roots of plants, the H+/K+ symporters are only one member of a group of
several symporters/antiporters that specifically allow only one charged hydrogen
ion (more commonly known as a proton) and one charged K+ ion. This group of
carriers all contribute to modulate the chemiosmotic potential inside the cell.

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Questions:
1. An example of cation transporter is .......... and an example of anion transporter is
........
2. The activity of antiports depends upon ...................
3. Glycerol-3-phosphate

transport

is a. symport
b.uniport
c. antiport
d. ATP dependent transport.
4. What are transporters and cotransporters?
5. Differentiate between symport and antiport.
6. Describe Na+ linked antiporters.
7. What are uniports?
8. Describe the mechanism of accumulation of metabolites and ions in plant
vacuoles.
9. What are the functions of cotransporters?
10. What are LacYsymporters?Describe their structure.

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References
+

1. Alper, S. L., M. N. Chernova, and A. K. Stewart(2001); Regulation of Na -

3-

independent Cl /HCO exchangers by pH,J. Pancreas2:171175.

2. J. M. Maathuis and D. Sanders (1992);Plant membrane transport, Curr. Opin. Cell
Biol. 4:661
3. Lewin (2011); Cells: Membranes and transport mechanism, 2

nd

edition

4. Lodish H, Berk A, Zipursky SL, W. H. Freeman (2000); Cotransport by

Symporters and Antiporters: Molecular Cell Biology; 4th edition.
5. M. Joanne Lemieux; Yafei Huang; Da Neng Wang (2005); Crystal structure and
mechanism of GlpT, the glycerol-3-phosphate transporter from E.coli, Journal of
Electron Microscopy 54 (Supplement 1): i43-i46
6. M. Lodish (2003); Molecular cell biology: Chapter 7 Transport of ions and small
th

molecules across cell membranes, 5 edition

+

7. P. Rea and D. Sanders (1987); Vacuolar H -translocatingpyrophosphatases: a new

category of ion translocase, Physiol. Plant 71:131

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Module 3 Lecture 6
Transport in prokaryotic cells
Transport in prokaryotic cells: The transport system of a cell depends upon the
substrate requirements of the cell, the bioavailability of the substrate and the
environmental conditions. It also depends on the metabolic features and physiological
state of the organism. Prokaryotic cells have simpler structure and mostly are unicellular.
Hence their transport system is different from higher eukaryotes. Here we will study the
transport in prokaryotic cells with respect to bacteria.
Membranes in bacteria: Membranes play a major role in transport. The different types
of membrane found in bacteria are:
1. Cytoplasmic membrane, in all bacteria
The inner membrane of a cell is different from outer membrane of a cell. And the space
between these membranes is called periplasam. The membrane is symmetrical, with an
equal distribution of lipids (exclusively phospholipids, mainly phosphatidylethanolamine,
phosphatidylglycerol and cardiolipin) among the inner and outer surface. Some of the
functions associated with cytoplasmic membrane which has role in transport mechanism
of cell are:
Osmotic and permeability barrier
Presence of transport system for various solutes
Synthesis of membrane lipids
Assembly and synthesis of extracytoplasmic proteins
Coordination of DNA replication and segregation with septum formation and cell
division
Energy generation functions such as electron transport system, establishment of
photon motive force and transmembrane ATP-synthesizing ATPase

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2. Outer membrane, mostly in gram negative bacteria

The outer membrane is highly asymmetrical, with the inner leaflet, oriented to the
periplasam. The outer leaflet, facing the external medium contains lipopolysaccharides
(LPS) constituting of three parts: lipid A as anchor, the core oligosaccharide functioning
as spacer element and an O-specific polysaccharide consisting of oligosaccharide
repeating unit. Proteins are the integral components or associated with OM. Some of the
functions associated with OM are:
Involved in transport mechanism.
Contribution of membrane integrity
Serves as anchor for flagellae, fimbriae and pili. Hence important for locomotion, cellcell interaction, adhesion to surfaces and formation of biofilms.
LPS are major antigenic determinants, preventing entry of cell-damaging components
and serve as receptor for a number of bacteriophages.
3. Cell walls of gram positive bacteria
The cell walls of gram positive bacteria are devoid of outer membrane but possess a thick
murein layer. In Gram-positive bacteria, teichoic acids are covalently linked to
peptidoglycan. Teichoic acids are polyol phosphate polymers with a strong charge. They
are strongly antigenic and absent in Gram-negative bacteria. In some species, teichuronic
acids are found as lipoteichonic acids which are composed of glycerol teichoic acid
linked to glycolipid. Additional wall components can be polysaccharides, lipids and
proteins.
4. Membrane that forms envelope in mycobacteria
Membrane that forms envelope in mycobacteria is characterized by their low
permeability, which contributes resistance of the microbes to therapeutic agents. It
contains two special features: an outer lipid barrier based on a monomer of mycolic acids
and a capsule-like coat of polysaccharide and protein. The cell wall contains a large
amount of C60-C90 fatty acids, mycolic acids that are covalently linked to arabinogalactan.

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Transport process:
Transport process can be divided into four classes on the basis of driving forces and
modes of energy coupling (Milton H. Saier et al., 2000):
1. Passive diffusion:
The passive diffusion occurs along the concentration gradient and without the use of
metabolic energy. Some solutes pass the permeability barrier of a lipid bilayer by passive
diffusion. This is valid for small apolar molecules and small slightly polar but uncharged
molecules like water and dissolved gases. Some other solutes are also transported via
channels or channel type proteins to overcome in a diffusion-controlled movement.
2. Primary active transport:
Primary active transport is characterized by coupling translocation of solute directly to a
chemical or photochemical reaction. Primary source includes pyrophosphate bond
+

hydrolysis, methyl transfer and decarboxylation. Transport of Na and K by carrier

+

protein, Na - K ATPase, is the most common example of primary active transport.

3. Secondary active transport:
In secondary active transport the translocation step across the membrane is coupled to the
electrochemical potential of a given solute. The solute chemical potential created by
primary active transport systems is the driving force, which allows an uphill transport of
another solute, against its own concentration gradient. The uptake can be mediated as
uniprot, symport and antiport. A common example of secondary active transport is the
+

symport of Na and glucose. The transmembrane protein Na - glucose transporter, acts

+

as a carrier, allows Na and glucose to enter the cell together. The Na flow down their
concentration gradient while the glucose molecules are transported against their
+

concentration gradient into the cell. Later the Na is pumped back out of the cell by the
+

Na -K ATPase.

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4. Phosphophenolpyruvate: sugar phosphotransferase system (Pts):

Pts translocation process is exclusive to bacterial species which phosphorylates its
carbohydrate substrates during transport.

Figure 1: Classification of major types of transport mechanism across biological membranes based on function and phylogeny

The major transport mechanism based on the mode of transport, energy coupling
mechanism and substrate specificity and protein phylogenetic grouping that reflects
structure, function and its mechanism are:
1. Transport independent diffusion
Gases (such as O2 and CO2); hydrophobic molecules (such as benzene) and small
polar but uncharged molecules (such as H2O and ethanol) are able to diffuse across the
plasma membrane.

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2. Transport dependent diffusion

This transport takes allows polar and charged molecules such as carbohydrates, amino
acids, nucleotides and ions, to cross the plasma membrane.
a. Channels
Some examples are voltage gated channels which open in response to change in
electric potentials; others called ligand gated channels open in response to the binding
of the ligand.
(i) -helical protein channel
(ii) -barrel proteins
(iii)Toxin channels
(iv) Peptide
channels b. Carriers
The common example is the movement of glucose mediated by carrier protein called
glucose transporter (GLUT).
(i) Primary active transport: Mechanically driven, Light absorption driven, Methyl
transfer driven, Oxidoreduction driven, Decarboxylation driven, Pyrophosphate
bond hydrolysis driven
(ii) Uniporters
(iii) Secondary active transport: Cation symporters, Cation antiporters, Solute solute
antiporters
(iv) Group translocators
Some examples of transporter in bacteria can be studied with the following
examples:
1. Phosphate transport:
Two major phosphate transport systems are involved in bacteria:
a. Low affinity Pit (phosphate inorganic transport) system
b. High affinity Pst (phosphate specific transport) system

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Pit consists of a single trans-membrane protein and is constitutively expressed secondary

transporter. This system is characterized by uptake of phosphate which is in the form of a
neutral metal phosphate complex and is in symport with a proton. This transport of
phosphate is achieved by binding and dissociation of the neutral metal phosphate
+

complex and H on the outer and inner surface of the trans-membrane protein carrier
protein. Pit is reversible and therefore allows both import and export of divalent ions and
phosphate. Also it has a relatively low specificity for both phosphate and arsenate (toxic
analogue of phosphate).

Figure 2: Phosphate transport by low affinity pit

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In contrast, protein specific transport (Pst) is a periplasmic protein-dependent transporter.

It consists of four subunits: a phosphate-binding protein located in the periplasmic space,
two cytoplasmic associated proteins that contain six membrane spanning helices and a
dimeric ATP binding protein. It operates as a primary transport mechanism i.e.
unidirectional phosphate transport is coupled to a chemical reaction. Phosphate is
-

transported in the form of H2PO4 and HPO4

in Pst system and has a relatively high

substrate affinity.

Figure 3: Phosphate transport by ATP dependent high affinity Pst system

Besides, phosphate also enters the cell in the form of esters such as sn-glycerol-3phosphate, glucose-6-phosphate or mannose-6-phosphate. Other organic phosphate
compounds may diffuse through the outer membrane before hydrolysis in the periplasm
by phosphatases - allowing transport of Pi into the cytoplasm. Pi linked antiport systems
of sn-glycerol-3-phosphate (GlpT) and glucose-6-phosphate (UhpT) mediate the
translocation of organo-phosphate compounds across the cell membrane. Phosphate is
also accepted as an analogue of organo-phosphate by these exchange systems; the affinity
for phosphate is lower than for the organo-phosphate. PhoE pores are formed in E. coli
cell membranes during phosphate limitation and have a preference for anions such as
phosphate and phosphate-containing nutrients, facilitating the unspecific entry of
phosphate into the cytoplasm by diffusion.

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2. Arsenic transport:
It was studied that auqaporins facilitate the diffusion of metalloids such as arsenic (As)
and antimony (Sb). The trivalent forms of these metalloids are structurally similar to
glycerol at neutral pH and hence enter cells through aquaporins.
3. Magnesium transport:
Transport of Mg

2+

into the cell is problematic, in spite of their largest hydrated radius,

2+

smallest ionic radius, and highest charge density. Transport systems for Mg have been
characterized well in Salmonella typhimurium. The CorA transport system is expressed
2+

constitutively and is the major Mg transporter in Eubacteria and Archaea. It consists of

three transmembrane domains, a large periplasmic domain, and no sequence homology to
other proteins. The MgtE Mg

2+

transporter also lacks sequence homology to other

proteins, and it is unclear if Mg

2+

transport is its primary function. The MgtA and MgtB

Mg

2+

transporters have sequence homology to P-type ATPases and closely related to the

mammalian Ca

2+

ATPases than to the prokaryotic P-type ATPases. Both transporters

2+

mediate Mg influx with, rather than against its electrochemical gradient. Unlike CorA
and MgtE, the MgtA and MgtC/MgtB loci are regulated, being induced by the twocomponent regulatory system PhoP/PhoQ. PhoQ is an Mg

2+

membrane sensor kinase that

2+

phosphorylates the transcription factor PhoP under Mg - limiting conditions. This factor
then induces transcription of MgtA and MgtCB.
4. In hyperthermophillic Archaea, only tranporters of ABC type are useful in uptake of
carbohydrates (e.g. glucose, cellobiose, maltotriose, arabinose, trahalose). This reflects an
adaptation to the extreme habit, enabling organisms to acquire all available sugars very
effectively.

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Interesting facts:

Transport system of a cell depends upon the substrate requirements of the cell, the
bioavailability of the substrate, environmental conditions and membrane
permeability.

Phosphate can be transported either by low affinity pit or ATP dependent high
affinity Pst system.

In spite of largest hydrated radius, smallest ionic radius, and highest charge
density of Mg

2+

, its transport into the cell is problematic.

Only tranporters of ABC type are useful in uptake of carbohydrates in

hyperthermophillic Archaea.

Questions:
1. Transport of solutes across cells depends upon:
a. Substrate requirements of the cell and bioavailability of the substrate.
b. Environmental conditions and membrane permeability.
c. Metabolic features and physiological state of the organism.
d. All of the above.
2. The type of transport without any energy input in the cell is called:
a. Passive transport
b. Active transport
c. Osmosis
d. Plasymolysis
e. Turgor pressure
3. Which of the following pieces of evidence would suggest that a substance entered
a cell via active transport as opposed to passive transport?
a. The substance moved from a high concentration to a low concentration.
b. ATP was required for transport.
c. The substance moved across the membrane via a carrier protein.
d. None of the above.

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4. What are the functions associated with cytoplasmic membrane which has role in
transport mechanism of cell?
5. What are the composition of outer membrane and its functions that has role in
transport mechanism of cell?
6. What is the classification of transport mechanism in the cells? Explain with
example.
7. Explain the transport mechanism of phosphate in the cell.
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