Bbcct-111 Membrane Biology and Bioenergetics

BBCCT-111 MEMBRANE BIOLOGY AND Indira Gandhi National Open University BIOENERGETICS School of Sciences

Block 2 MEMBRANE TRANSPORT UNIT 4 Membrane Transport-I 55 UNIT 5 Membrane Transport-II 71 UNIT 6 Vesicular Transport and Membrane Fusion 85 Block 1 Biomembranes ...... BLOCK 2: MEMBRANE TRANSPORT

Protein mediated transport is specific and incredibly diverse. In this Block on Membrane Transport we shall explore the different transport mechanisms, be it transport of small molecules through transport protein, ions, water molecules or common metabolites like glucose or amino acids. Unit 4 deals with simple and facilitated diffusion, and different forms of active transport. Group translocation which deals with transport of biological macromolecules through relatively narrow pores across biomembranes is discussed in Unit 5. This unit also covers details about voltage and ligand gated ion channels. Another mode of exchange based on vesicular transport is covered in Unit 6. Vesicular transport is a mode of exchange across membranes mechanistically different from transport of small molecules and ions and group translocation. Fusion and fission of membrane play significant role, which itself is regulated by membrane proteins. All these aspects are discussed in detail in this block. Expected Learning Outcomes

After studying this block, you should be able to:

x Discuss transport across membrane and explain the role of transport proteins in mediating the facilitated diffusion and active transport;

x Explain various types of ion channel proteins; x Describe the ABC family of transporters; x Differentiate between clathrin and COP coated vesicles; and

x Explain membrane fusion and receptor mediated endocytosis. We hope that after studying this block you will acquire understanding of the fundamentals of different transport mechanisms across biological membranes. Wishing you success in this endeavour !!

54 Unit 1 Introduction to Biomembranes ......

UNIT 4

MEMBRANE TRANSPORT-I

Structure 4.1 Introduction 4.4 Cytosis

Expected Learning Outcomes 4.5 Summary

4.2 Simple and Facilitated Diffusion 4.6 Terminal Questions

Simple Diffusion 4.7 Answers Facilitated Diffusion 4.8 Suggested Readings Osmosis

4.3 Active and Passive Transport

Active Transport Passive Transport

The FRAP does not distinguish the protein that are immobilized or can only diffuse over a limited distance in a 4.1 INTRODUCTION given time. The alternative technique, In the previous block, you studied about the structural aspects of biological SPT (Single Particle Tracking) can overcome membrane. Now you know in detail about membrane composition and fluid such hurdles. In SPT, mosaic model. You have also studied some of the characteristic features like individual protein membrane asymmetry, membrane fluidity, membrane dynamics as well as molecule is labeled with techniques like freeze fracture and FRAP (Fluorescence Recovery after antibody coated with gold particles. The Photobleaching) which are employed to study membrane and its properties. In movement of the labeled this unit you will learn about one of the functional aspects of biomembrane. molecule is followed by Apart from providing barrier and protection from the external environment, Computer Enhanced biomembrane plays a crucial role in transport of substances, ions, molecules Video Microscopy. and drugs across biological membranes. 55 Block 12 MembBiraneomem Transporbranest ...... Membrane transport is essential for the life of a cell. At cellular level vast amount of exchange is necessary to maintain its structure and function. The transport may involve the addition of biological molecules and the discharge of waste products. Membrane transport refers to the movement of particles (solute and ions) across or through the phospholipid bilayer membrane. It depends upon the permeability of the membrane, transmembrane concentration difference and the size and charge of the solute. Transport across membrane may be by passive, facilitated or active mechanisms. Some of these mechanisms require the expenditure of energy and use of transmembrane proteins. We will begin the unit with simple phenomenon like diffusion and go into the details of active and passive transport. We will also focus on some ATP powered pumps as an example to understand their mode of functioning.

Expected Learning Outcomes

After studying this unit, you should be able to:

describe the process of diffusion;

differentiate between simple and facilitated diffusion;

describe active and passive transport;

differentiate between active and passive transport;

describe various types of transport mechanisms; and

explain various types of ion channel proteins. 4.2 SIMPLE AND FACILITATED DIFFUSION

Biological membranes are composed of lipids and proteins. The lipid bilayer serves as a barrier to the movement of hydrophilic (water soluble) molecules across the membrane. However, it selectively allows the passage of selected ions and molecules which contribute to the steady state condition required for The concentration biological processes. Generally, the proteins embedded in the lipid bilayer difference of a molecule ion across the two sides function as transporter or receptor of extracellular stimuli. of a membrane is Let us recall the structural features of biological membranes which you have termed as gradient. studied in Unit 1. Biological membranes are composed of lipids and proteins. Movement from a high The major lipid in membranes is phospholipid where two fatty acid chains are concentration to a low attached to OH of the 1st and 2nd carbon of glycerol. The OH on the 3rd carbon concentration is known as movement with or in is attached to a polar organic alcoholic molecule and phosphate group. These the direction of side chains make one end as polar and the other end as non-polar in the concentration phospholipid molecule. In the lipid bilayer, the polar part is outside and non- gradient or downhill. polar part faces inside. The non-polar interior prevents the passage of water Movement from a low concentration to a high soluble substances through the bilayer. The ability of the lipid bilayer to concentration is known discriminate between low and high molecular weight hydrophilic molecules, as movement against permitting the former to diffuse across but not the latter is due to the presence concentration of pores in the bilayer. These pores are formed randomly as a result of thermal gradient or uphill. movement of acyl phospholipid chains. Mostly, the globular proteins in the membrane are inserted in the bilayer with 56 Unit 14 IntroductiMonem tborane Biomem Transporbranet-sI ...... their non-polar portion in contact with lipid and polar portion emerging out from the membrane surface. These proteins serve as channels for specific ions or molecules to cross this hydrophobic barrier, since many molecules required by the cell are polar and cannot enter freely into the cell. These polar molecules enter the cell through specific channel proteins found in the plasma membrane. The channels are specific transmembrane proteins having polar amino acids in the interior area. On the other hand hydrophobic non polar molecules can enter or leave freely the cell through lipid bilayer. This spontaneous process is termed diffusion. This process allows the small and hydrophobic (or lipophilic) molecule to move inside and outside the cell membrane. Diffusion is movement of substances (atoms, ions, molecules) from a region of higher concentration to a region of lower concentration. This results in uniform distribution of the substance. The driving force for diffusion is concentration gradient. Diffusion obeys Fick’s law, which states that the rate of movement of a substance is directly proportional to the concentration gradient. Diffusion can be of three types:

x Simple diffusion x Facilitated diffusion

x Osmosis (in aqueous medium) 4.2.1 Simple diffusion

Smaller molecules and nonpolar substances move freely through lipid bilayer from the side of higher concentration to lower concentration. This type of movement is shown by oxygen, nitrogen, benzene, water, urea, glycerol, carbon dioxide etc. The rate of diffusion is directly proportional to the concentration difference, surface area, distance, and temperature and inversely proportional to the size of molecule. Simple diffusion thus shows a linear relationship between the concentration of solute and its rate of transport across the membrane. All water-soluble molecules or ions that enter or leave the cell are either transported by carriers or pass through channels. Ions and some other polar molecules diffuse across the cell membrane through channels. Ion channels are proteins containing a hydrated interior portion that spans throughout the bilayer. Ions diffuse through the channel in either direction without meeting the lipid bilayer. Furthermore, these ions do not interact or bind to the channel proteins. The diffusion through ion channels is an essential component of signaling in the nervous system. Some of the molecules and their permeability across the plasma membrane are given in Table 4.1. 4.2.2 Facilitated diffusion The diffusion of some ions and other solutes like sugars and amino acids across the membrane is facilitated by carriers. Carriers bind their substrates with high stereospecificity and undergo cyclic conformational changes while channels do not do so. Channels are less specific to substrates but facilitate faster transport. In this type of transport the molecule binds physically to the carrier protein on one side of the membrane and is released on the other side (Fig. 4.1). This movement is from a higher concentration side to the lower 57 Block 12 MembBiraneomem Transporbranest ...... concentration side. The cell uptakes the required essential molecules like sugars and prevent the accumulation of unwanted molecules by facilitated diffusion. However, the rate of movement through channel proteins is much higher than transport through carrier proteins. Membrane proteins that speed the movement of a solute across the membrane by facilitating diffusion are called transporters or permeases.

Table 4.1: Examples of some molecules and their permeability across the plasma membran

S. Category Molecules Permeability No.

1. Gases CO2, O2, N2 Permeable

2. Small uncharged polar Ethanol Permeable molecules Urea, Water Slightly Permeable

3. Large uncharged polar Glucose, Fructose Impermeable molecules

4. Ions K+, Mg2+,Ca2+, Cl-, Impermeable - 2- HCO3 , HPO4

5. Charged polar Amino acids, ATP, Impermeable molecules Glucose-6-phosphate, Proteins, Nucleic acids

In hypertonic solutions, there are more solute molecules outside the cell which causes the water molecules to be sucked out of the cell and the cell shrinks. In hypotonic solutions, there is less solute molecules outside the Fig. 4.1: Overview of membrane transport proteins. Passive transport across ion cell, water will move channels or carriers is driven by a chemical gradient. Active transport by carrier inside the cell. The cell proteins/ transporters allows a transport against a chemical gradient, thanks to will grow larger and the consumption of energy, e.g. by ATP hydrolysis. finally burst. 4.2.3 Osmosis In isotonic solutions, there is equal Osmosis is a special example of diffusion. It is diffusion of water molecule concentration of solute on both side of the through semipermeable membranes from a dilute solution to a concentrated membrane; the solution. This process does not require any energy. Absorption of water by movement rate of water plant roots is an example of osmosis in plant. Plant cells in a hypotonic will be equal in both the solution gain water by osmosis and the plant tissue becomes stiffer (turgid) directions. while in a hypertonic solution plants loose water by osmosis and tissue 58 becomes flaccid (shrunken). Unit 14 IntroductiMonem tborane Biomem Transporbranet-sI ...... Let us understand some of the terms which you will come across frequently in this block.

Channels: Channels are proteins having several helical segments that are spread back and forth through the membrane, forming a channel. For example, the bacteriorhodopsin contains seven nonpolar helical segments that traverse the membrane, forming a circular pore through which protons pass during the light-driven pumping of protons.

Pores: Some transmembrane proteins have extensive nonpolar regions with secondary configurations of E-pleated sheets instead of D helices. The E sheets form a characteristic motif, folding back and forth in a circle so the sheets come to be arranged like the staves/poles of a barrel. This E- barrel, open on both the ends, is a common feature of the porin class of proteins that are found within the outer membrane of some bacteria.

Transmembrane proteins: Cells contain a variety of different transmembrane proteins, which differ in the way they traverse the bilayer, depending on their functions. They are anchored into the bilayer by their nonpolar segments. While anchor proteins may pass through the bilayer only once, many channels and pores are created by proteins that pass back and forth through the bilayer repeatedly, creating a circular hole in the bilayer.

Tick [9] mark the correct option: a) Biological membrane provide barrier to hydrophobic molecules. [True/False] b) Glycerol is a three carbon molecule. [True/False] c) A phospholipid contains three fatty acid chains. [True/False]

d) Active transport does not require energy. [True/False] e) Active transport is required for concentrating metabolites in a particular organelle of the cell. [True/False]

Fill in the blanks with appropriate words: a) Polar molecule enters the cell through specific …………. (channels/ anchors) in the plasma membrane. b) Osmosis is the phenomenon of diffusion of water molecules through …………... (permeable/semipermeable) membrane from a dilute solution to a more …………… (concentrated/diluted) solution.

c) The interior side of plasma membrane is ……………. (positively/ negatively) charged relative to the extracellular side of the membrane.

59 Block 12 MembBiraneomem Transporbranest ...... 4.3 ACTIVE AND PASSIVE TRANSPORT

Transport of substances against their concentration gradient (from lower concentration to higher concentration) is energetically unfavorable; therefore energy is required for this process. However, this process is essential for:

i) Maintaining the balance of ions across the membrane

ii) Concentrating metabolites in a particular organ of cellular compartment

iii) Exporting foreign/ toxic substances from the cell

As mentioned in the previous section, carrier proteins and channels are two major classes of membrane transport proteins. The transport through these proteins can be further classified into two types depending on the requirement of energy for the transport process:

i) Active transport

ii) Passive transport 4.3.1 Active Transport

It is further classified into two types: primary active transport (PAT) and secondary active transport (SAT). Primary active transport uses the energy stored in ATP, photons and electrochemical gradients directly for the transport of molecules from low concentration areas to high concentration area across the cellular membrane. The PAT is facilitated by P-class pump, V-class pump, F-class pump and ABC transporters. Let us discuss briefly, the main energy sources driving PAT. i) ATP hydrolysis: The enzyme catalyzed hydrolysis of ATP forming ADP induces a conformational change in the transport protein facilitating the influx or efflux of particles. The enzyme catalyzing this process is known as ATPase. The most common example of ATP hydrolysis during primary active transport in cells is Na+/ K+ pump. ii) Electrochemical gradient energy: An electrochemical gradient has two components: (1). An electric component resulting from difference in charge on either side of the membrane and (2). A chemical component resulting from the difference in concentration of ions across the cellular membrane. The electrochemical gradient is generated in terms of the presence of proton (H+) gradient. iii) Photon energy: The energy stored in a photon, is used to generate a proton gradient. The stepwise movement of electrons in an electron transport chain reduces NADPH and subsequently generates proton gradient. In secondary active transport the movement of molecules is from lower concentration to higher concentration utilizing energy. However, in this case the direct hydrolysis of ATP does not occur. Instead, the electrochemical gradient generated from pumping of ions out of the cell is used. Secondary active transport is further classified as uniport, symport or antiport (Fig. 4.2).

60 i) Uniport: Uniporters transport a single type of molecules/ions down its Unit 14 IntroductiMonem tborane Biomem Transporbranet-sI ...... concentration gradient via facilitated diffusion. For example, glucose and amino acids cross the plasma membrane in most mammalian cells with the help of uniporters.

ii) Symport: When the carrier protein transports two solutes in the same direction, it is called symport. It uses a downhill movement of one molecule/ion to transport other molecule against its concentration gradient. Symporters move both the molecules or ions in the same direction through a transmembrane transport protein. They usually have opposite charge.

iii) Antiport: When an ion traverses in one direction and simultaneously another metabolite is transported in the other direction, then it is called antiport. It moves two or more different molecules/ions across the cellular membrane in opposite directions. Secondary active transporter antiport protein moves one molecule/ion down its concentration gradient and uses the energy produced from this process for the movement of another molecule/ion up its concentration gradient. This is similar to symport mechanism. The only difference being instead of moving in same direction molecules move in opposite direction.

Fig. 4.2: Overview of transporters. Now, let us focus our attention on a major class of proteins, the ATP-powered pumps, that use the energy released by hydrolysis of the terminal phosphoanhydride bond of ATP to transport ions and various small molecules across membranes against their concentration gradients. Although these proteins are commonly called ATPases, they normally do not hydrolyze

ATP into ADP and Pi unless ions or other molecules are simultaneously transported. For example Na+- K+ pump (also known as Na+- K+ ATPase), Ca++- ATPase, H+- K+ATPase etc. These ATP driven ion pumps and transporters utilize ATP for uphill transport whereas in case of coupled transport, the energy stored in ion gradients is used. Coupled transporters are also known as secondary active transporters, such as symporter & antiporters.

ATP-Powered Pumps All ATP-powered pumps are transmembrane proteins with one or more binding sites for ATP located on subunits or segments of the protein that face the cytosol. These pumps are grouped into four classes. Three of the classes P-type, F-type, and V-type only transport ions, whereas members of the ABC family primarily transport small molecules such as amino acids and sugars. Here you must note that all of these ATP powered pumps are involved in primary active transport as mentioned in the beginning of this section. 61 Block 12 MembBiraneomem Transporbranest ...... Na+-K+ ATPase in the plasma membrane of animal cells and Ca2+ ATPase pumps are examples of P-type of ATP powered pumps. During transport, at least one of the subunits is phosphorylated (hence the name “P” class), and the transported ions move through the phosphorylated subunit of the pump. All known V and F pumps transport only protons. The H+ pump that generates and maintains the plasma membrane electric potential in plant, fungal, and bacterial cells, also belong to V-type. F-class pumps are found in bacterial plasma membranes and in mitochondria and chloroplasts. In contrast to V- class pumps, F-class pump generally function as a kind of reverse proton pump. The energy released by the movement of protons form the exoplasmic to the cytosolic face of the membrane down the proton electrochemical

gradient is used to power the synthesis of ATP from ADP and Pi. Because of their importance in ATP synthesis in chloroplasts and mitochondria, F-class proton pumps are commonly called ATP synthases. ABC (ATP-binding cassette) is a large family of multiple members that are more diverse in function, for example multidrug-resistance proteins. You will study about ABC family of transporters in detail in next unit.

Have you ever wondered why do we need so many diverse transporters? Just think about the ionic composition. The ion composition inside the cell is different from that in the extracellular fluid, and this difference has to be maintained for cell survival. For example the gradients of sodium (Na+), calcium (Ca2+), potassium (K+) and chloride (Cl-), must be maintained within proper limit to ensure normal functioning of the cell and maintenance of the membrane potential. In fact, all plasma membranes have electric potentials (transmembrane potential) across them with inside of the cell being more negative compared to the outside. This is due to active transport of ions particularly H+ ions out of the cell. This potential difference allows for the entry of positively charged ions into the cell but opposes the entry of negatively charged ions. For example, the sodium ion (Na+) has higher concentration outside of the cell than inside. This is maintained by a constant active transport of Na+ out of the cell, which is also helpful in maintaining the osmotic pressure on both the sides of the membrane. Ca2+ and Cl- concentrations are also higher outside the cell than cell interior. On the other hand, K+ is higher inside than the extracellular concentrations. This difference in the concentration gradient leads to a change in electrochemical gradient across the cell membrane. Table 4.2 gives the concentration of some of the commonly occurring ions across the biological membrane. Table 4.2: Concentration of some important physiological ions across cell membrane.

S. No. Ion Concentration in Concentration in cytosol (mM) extracellular space (mM)

1Na+ 15 145

2K+ 120 4.5

3Cl- 20 116

2+ -4 62 4Ca10 1.2 Unit 14 IntroductiMonem tborane Biomem Transporbranet-sI ...... The combined effect of electrical and concentration gradients are termed as electrochemical gradient. In case of K+ and Cl-, the electrical and chemical gradients of molecules work against each other. K+ is attracted towards the inside of the cell because of the interior negative membrane potential, but its concentration gradient works in opposite direction. Thus K+ reaches almost to equilibrium across the plasma membrane and tries to move out of the cell. Cl- also tends to move outside of the cell because of the negative interior membrane potential although its concentration outside is much higher. In case of Na+ and Ca2+, both tend to come inside the cell down the concentration gradient and the negative membrane interior pulls them inside the cell. So, let us try to understand how these pumps work in order to maintain the required gradient.

Sodium- Potassium Pump

More than one third of all of the energy exhausted by a non-dividing animal cell is utilized for the active transport of sodium (Na+) and potassium (K+) ions. Most animal cells have a low internal concentration of Na+, relative to their surroundings, and a high internal concentration of K+. They maintain these concentration differences by actively pumping Na+ out of the cell and K+ in by the sodium-potassium pump. The cell obtains the energy it needs to operate the pump from hydrolysis of adenosine triphosphate (ATP).

The sodium-potassium pump is an active transport process, transporting Na+ and K+ from areas of low concentration to the areas of high concentration. The sodium-potassium pump works through a series of conformational changes in the trans-membrane protein. The main steps of the process are as follows: 1. Three sodium ions bind to the cytoplasmic side of the pump leading to change in its conformation. 2. In this new conformation, the protein binds a molecule of ATP and cleaves

it into adenosine diphosphate and phosphate (ADP + Pi). ADP is released; but the phosphate group remains bound to the protein. The protein is now in the phosphorylated state.

3. The phosphorylation of the protein induces a second conformational change in the protein. This change translocates three Na+ across the membrane, so they now face the exterior. In this new conformation, the protein has a low affinity for Na+, and the bound Na+ dissociate from the protein and diffuse into the extracellular fluid.

4. However, the new conformation has a high affinity for K+, two of which bind to the extracellular side of the protein as soon as the Na+ is released.

5. The binding of the K+ causes another conformational change in the protein, and the bound phosphate group is released.

6. De-phosphorylated protein reverts back to its original conformation, exposing the two K+ to the cytoplasm and releasing them into the interior of the cell. The original conformation has a high affinity for Na+; when these ions bind, they initiate another cycle. Three Na+ ions leave the cell and two K+ ions enter inside every cycle. The changes in protein conformation that occur during the cycle are rapid, 63 Block 12 MembBiraneomem Transporbranest ...... enabling each carrier to operate about 100 cycles and transporting as many as 300 Na+ per second.

Proton Pump

The proton pump pumps protons (H+ ions) across a membrane using energy derived from energy-rich molecules or from photosynthesis, for example NADH dehydrogenase, cytochromes c oxidase etc. This generates a proton gradient across the membrane. Generally, membranes are impermeable to protons; therefore protons can diffuse back down their concentration gradient by another co-transport protein like ATP synthase. You will learn about the functions of these transport proteins in next block. The movement of protons through their co-transport protein is coupled to the production of ATP, the energy-storing molecule. 4.3.2 Passive Transport

Through the channels, the substances simply diffuse down the concentration gradient and usually do not require energy. This is called as passive transport. Small hydrophobic molecules are able to cross membrane by simple diffusion.

Na+- Glucose Symporter

Many amino acids and sugars are accumulated in animal cells against their concentration gradient i.e. they are transported into the cell from the extracellular fluid, even though their concentrations are higher inside the cell. The transport of these molecules is coupled with sodium ions to enter the cell down the Na+ concentration gradient established by the sodium-potassium pump. Thus, Na+ and a specific sugar or amino acid simultaneously binds to the same transmembrane protein, on the outside of the cell and is an example of symport. Both are then translocated to the inside of the cell, but in the process Na+ moves down its concentration gradient while the sugar or amino acid moves against its concentration gradient. The cell uses some of the energy stored in the Na+ concentration gradient, to accumulate sugars and amino acids.

The Na+ - driven glucose pump is an example of symporter. It is found on the apical side of intestinal epithelial cells. After a meal when the concentration of sugar is higher inside the cell than outside, this pump transports glucose into gut epithelial cells. This co-transporter is dependent on Na+ gradient for its energy which flows into the cell down its gradient to allow the transport of glucose into the cell.

Na+-Ca2+ Antiporter

In a related however different process, called counter-transport, the inward movement of Na+ is coupled with the outward movement of another substance, such as Ca++ or H+. Similar to co-transport, both Na+ and the other substance bind to the same transport protein, in this case called an antiport, but during antiport they bind on opposite sides of the membrane and are moved in opposite directions. In counter-transport, the cell uses the energy released from Na+ movement down its concentration gradient into the cell to extrude a substance up its concentration gradient. 64 Unit 14 IntroductiMonem tborane Biomem Transporbranet-sI ...... The Na+/Ca2+ exchanger also uses the electrochemical gradient of Na+ to allow the transport of Ca2+. In this case, Na+ flows into the cell down its concentration gradient, which allows the export of Ca2+ from the cell against its gradient. Three Na+ molecules enter the cell for exit of each Ca2+.

Anion Transporter

- Bicarbonate (HCO3 ) transport mechanisms are the principal regulators of pH in animal cells. Such transport also plays a vital role in acid-base maintenance in the stomach, pancreas, intestine, kidney, reproductive organs and the - - central nervous system. For example, exchange of HCO3 for Cl in a + - reversible, electroneutral manner or Na /HCO3 co-transport where proteins + - mediate the coupled movement of Na and HCO3 across plasma membranes, often in an electrogenic manner.

Glucose Transporter

Several glucose transporters mediate the thermodynamically downhill movement of glucose across the plasma membranes of animal cells. These transporters are named as GLUT1 to GLUT5 and have distinct roles to perform which is given briefly in Table 4.3. These transport proteins are made up of about 500 residues long amino acids and possess a common 12 transmembrane helix structure. Table 4.3: Glucose transporters and their functions Name Location Function

GLUT1 All mammalian tissues Basal glucose uptake

GLUT2 Liver and pancreatic cells In liver, GLUT2 removes excess glucose from the blood while in pancreas, it adjusts the rate of insulin secretion which accordingly signals the need for removal of glucose from the blood for storage as glycogen or conversion into fat

GLUT3 All mammalian tissues Basal glucose uptake

GLUT4 Muscle and fat cells Amount in muscle plasma Mostly, the inner membrane increases with membranes in a double endurance training membrane system are intrinsically GLUT5 Small intestine Primarily a fructose transporter impermeable to nearly all ions and polar Porins molecules. For instance, a large Porins are proteins found in the outer membranes of many Gram-negative number of transporters bacteria. They function to form a water-filled pore through the membrane, from are required to shuttle metabolites such as the exterior to the periplasm, which is a region located between the outer and ATP, pyruvate, and inner membranes. The porin channel allows the diffusion of small hydrophilic citrate across the inner molecules. mitochondrial membrane. Similarly, mitochondria also have two membrane systems, an outer and inner membrane. The outer membrane is quite permeable to most small molecules 65 Block 12 MembBiraneomem Transporbranest ...... and ions because of the presence of mitochondrial porin, a 30-35 kd pore forming protein also known as voltage-dependent anion channel (VDAC). This regulates the transport of usually anionic metabolites such as phosphate, chloride, organic anions, and the adenine nucleotides across the outer membrane. VDAC appears to form an open E-barrel structure similar to that of the bacterial porins. The outer membrane of bacteria, like that of mitochondria, is permeable to most small metabolites because of the presence of porins. Beta-based membrane potein is found in the 4.4 CYTOSIS outer membranes of bacteria, mitochondria Mechanism for transport of large quantities of molecules in and out of the cell and chloroplasts. They is termed as cytosis. It is broadly divided into two types: endocytosis and resemble barrels used to contain liquids. These E- exocytosis. barrel proteins serve essential functions in Endocytosis cargo transport, Most of the substances used as fuel molecules are polar and cannot move signaling and re also vital for membrane freely inside the lipid bilayer. The process used by single celled eukaryotes to biogenesis. internalize these substances is known as endocytosis, in which the plasma membrane extends outward and envelops the food particles. Endocytosis is further divided into three types: i) Phagocytosis ii) Pinocytosis iii) Receptor-mediated endocytosis

Phagocytosis is the process by which a cell takes in/ingests or engulfs large particles, microorganisms, dead cells or some fragment of big organic molecule. For example, amoeba surrounds the target objects with pseudopods for phagocytosis while white blood cells and macrophages engulf and kill foreign invading pathogens. Pinocytosis is common among eukaryotes. It is a process by which cell ingests the fluid by the inward folding of the plasma membrane and the formation of membrane-bound, fluid-filled vesicles (pinocytic/pinocytotic vesicles) either clatherin coated or caveolae derived. This is also known as cell-drinking. Look at (Fig. 4.3) and note the difference between the two processes.

(a) (b)

66 Fig. 4.3: Diagrammatic representation of phagocytosis and pinocytosis. Unit 14 IntroductiMonem tborane Biomem Transporbranet-sI ...... Receptor-mediated endocytosis is a specialized form of endocytosis involving specific receptor molecules. The molecule to be transported binds to the receptors on the plasma membrane. The specificity of this type of transport is provided by the structure of receptor in which only specific molecules can fit into. The interior portion of the receptor molecules resembles a hook that is trapped in an indented pit coated with the protein clathrin. The binding of molecules to the receptor initiates the endocytosis process (Fig. 4.4). The process is highly specific and very fast. Low density lipoprotein (LDL) is taken up by receptor mediated endocytosis. The LDL molecules bring cholesterol into the cell which is required for synthesis of membranes.

Fig. 4.4: Diagrammatic representation of receptor-mediated endocytosis.

Exocytosis The discharge of larger material from vesicles at the cell surface is termed as exocytosis. In plant cells, exocytosis helps in exporting materials required for cell wall synthesis through the plasma membrane. In animal cells, exocytosis provides a mechanism for secreting many hormones, neurotransmitters, digestive enzymes and other substances.

Fill in the blanks: a) When a carrier protein transport two solute in same direction, it is called …………. .

b) The Na+ driven glucose pump is an example of ………….. . c) The discharge of large material from vesicles at the cell surface is termed as ………….. . 67 Block 12 MembBiraneomem Transporbranest ......

Tick [9] mark the correct option:

a) One molecule of Na+ enters for the exit of each molecule of Ca2+. [True/False]

b) Water is secreted out by the process of pinocytosis. [True/False]

c) Low density lipoproteins (LDL) is taken up by receptor mediated endocytosis. [True/False]

d) Na+/ Ca2+exchanger is an example of antiport. [True/False]

4.5 SUMMARY

Let us recapitulate what we have learnt so far:

x The biological membranes allow the passage of selected ions and molecules which contribute to the steady state condition required for biological processes.

x Diffusion is the movement of substances (atoms, ions, molecules) from a region of higher concentration to a region of lower concentration.

x Osmosis is diffusion of water molecule through semipermeable membranes from a more dilute solution to a more concentrated solution.

x Transport of substances against its concentration gradient (from lower concentration to higher concentration) is energetically unfavorable, therefore requires energy and is known as active transport.

x When the carrier protein transports two solutes in the same direction, it is called symport. When ion traverses in one direction and simultaneously another metabolite is transported in the other direction, then it is called antiport.

x Endocytosis is the process to internalize the substances, in which the plasma membrane extends outward and envelops the food particles.

x The discharge of larger material from vesicles at the cell surface is termed as exocytosis. 4.6 TERMINAL QUESTIONS

1. What is passive transport?

2. Explain the process of simple diffusion. 3. How facilitated diffusion is different from active transport? 4. Describe various types of ATP powered pumps?

5. Write a note on Na+-K+ pump. 6. Differentiate between phagocytosis and pinocytosis. 7. What is endocytosis? Explain. 68 Unit 14 IntroductiMonem tborane Biomem Transporbranet-sI ...... 4.7 ANSWERS Self-Assessment Questions

1. a) false b) true c) false d) false e) true

2. a) channels b) semi permeable, concentrated c) negatively

3. a) symport b) symporter c) exocytosis

4. a) false b) false c) true d) false Terminal Questions

1. Passive transport is the simplest method of transport and is dependent upon the concentration gradient and size and charge of the solute. In passive transport, small uncharged particles move across the membrane until the concentration becomes same on both the sides of the membrane. Passive transport is independent of membrane proteins and does not require expenditure of energy. 2. In simple diffusion, small uncharged molecules of non-polar hydrophobic molecules pass through the lipid bilayer to leave or enter the cell, moving from areas of higher concentration towards areas of low concentration. For example, oxygen, carbon dioxide and most lipids enter and leave cells by simple diffusion. 3. Facilitated diffusion is a form of passive transport mediated by transport proteins situated within the plasma membrane. Facilitated diffusion allows the movement of hydrophilic molecule through the lipid bilayer. The movement is from high concentration towards lower concentration to achieve the equilibrium. Facilitated diffusion utilizes channel proteins to facilitate solute movement while active transport is the movement of particles through a protein from low concentration area to a high concentration area utilizing the energy of ATP hydrolysis. ATP hydrolysis induces a conformational change in the transport protein which facilitates the mechanical movement of the molecule. Active transport systems are energy coupled devices as chemical and mechanical processes are involved in the movement of the molecules and require energy.

4. ATP-powered pumps are transmembrane proteins with one or more binding sites for ATP located on subunits or segments of the protein that face the cytosol. These pumps are grouped into four classes. Three of the classes P-type, F-type, and V-type only transport ions, whereas members of the ABC family primarily transport small molecules such as amino acids and sugars. Na+/K+ ATPase in the plasma membrane of animal cells and Ca2+ ATPase pump are examples of P-type of ATP powered pumps. During transport, at least one of the subunits is phosphorylated (hence the name “P” class), and the transported ions move through the phsophorylated subunit of the pump. All known V and F pumps transport only protons. The H+ pump that generates and maintains the plasma membrane electric potential in plant, fungal, and bacterial cells also belongs to V-type. F-class pumps are found in bacterial plasma membranes and in mitochondria and chloroplasts. F-type pump, use the 69 Block 12 MembBiraneomem Transporbranest ...... energy released by the movement of protons to power the synthesis of

ATP from ADP and Pi (Refer section 4.3.1). 5. One of the most important pumps in animals cells is the sodium- potassium pump (Na+-K+ ATPase), which maintains the electrochemical gradient (and the correct concentrations of Na+ and K+) in living cells. The sodium-potassium pump moves two K+ into the cell while moving three Na+ out of the cell (Refer section 4.3.1). 6. Phagocytosis is the process by which a cell ingests or engulfs another organism or some fragment of big organic molecule while pinocytosis is a process by which cell ingests the fluid by the inward folding of the plasma membrane and the formation of membrane-bound, fluid-filled vesicles. This is also known as cell-drinking.

7. Endocytosis is the process in which large molecules enter into a cell. In this a small piece of cell membrane covers the particles and in entrapped and brought into the cell. If the particle is solid, it is called phagocytosis and if it is liquid droplet, the process is termed as pinocytosis. Sometimes specific receptors are involved for the process of endocytosis, then the process is known as receptor mediated endocytosis. For more details refer section 4.4. 4.8 SUGGESTED READINGS

x Garret, R.H., Grisham, C.M. (2016). Biochemistry (6th ed.). Boston, Cengage Learning. ISBN-10: 1133106293, ISBN-13: 978-1133106296

x Berg, J.M., Tymoczko, J.L. and Stryer L., (2012) W.H. Biochemistry (7th ed.), Freeman and Company (New York), ISBN:10: 1-4292-2936-5, ISBN:13:978-1-4292-2936-4.

x Nelson, D.L., Cox, M.M. (2017). Lehninger: Principles of Biochemistry (7th ed.). New York, WH: Freeman and Company. ISBN: 13: 978-1-4641-2611- 6 / ISBN:10:1-6412611-9.

x Lodish, H., Berk, A., Kaiser, C.A., Krieger, M., Bretscher, A., Ploegh, H., Amon, A., Scott, M.P. (2016). Molecular Cell Biology (8th ed.). New York, WH: Freeman & Company. ISBN-13: 978-1-4641-0981-2.

Voet, D.J., Voet, J.G., Pratt, C.W. (2008). Principles of Biochemistry (3rd ed.). New York, John Wiley & Sons, Inc. ISBN:13: 978-0470-

70 Unit 1 Introduction to Biomembranes ......

UNIT 5

MEMBRANE TRANSPORT-II

Structure 4.1 Introduction 5.5 Ionophores

Expected Learning Outcomes Valinomycin

5.2 ABC Transporters Gramicidin

Multidrug Resistance (MDR) Protein 5.6 Summary

5.3 Group Translocation 5.7 Terminal Questions 5.4 Ion Channels 5.8 Answers

Voltage-gated ion channels 5.8 Suggested Readings Ligand-gated ion channels Mechano-gated channels

5.1 INTRODUCTION

In Unit 4, you studied about the simple and facilitated diffusion as well as active and passive transport. It has been explained that various carriers and membrane transport proteins allow the movement of ions or molecules through semipermeable or selectively permeable biological membrane. In this unit you will learn more about transporters like ABC transporter with emphasis on multidrug resistance (MDR) protein, group translocation which is also an active transport process involving phosphorylation and ion channels. In the section on ion channels we will focus on gated ion channels, viz. voltage-gated and ligand-gated ion channels and look into some specific examples of both. In the end you will learn about ionophores which also act as mobile carriers across the membranes. 71 Block 12 MembBiraneomem Transporbranest ...... Expected Learning Outcomes After studying this unit, you should be able to:

describe the ABC family of transporter;

discuss multi drug resistance;

explain the significance of group translocation;

classify ion channels; and

explain the significance of ionophores in transport. 5.2 ABC FAMILY OF TRANSPORTERS

In the previous unit you learnt about the pumps which are transmembrane proteins capable of transporting ions/molecules across the cell membrane, mitochondria or other cell organelle. These ATP dependent pumps also known as ATPas e are the pumps that transport ions against their concentration The human genome gradients. In this section you will learn about the multidrug resistance (MDR) contains at least 46 protein which is an important example of ABC transporter. The MDR protein genes that encode ABC pumps hydrophobic drugs from cytosol to outside of the cell. These transporters, many of transporters play important role in removing harmful substances (like drugs which are involved in maintaining the lipid and other toxins) from the cells. bilayer and in Recently discovered, ABCs are found in many families of transport proteins. transporting sterols, sterol derivatives, and ABC stands for ATP- binding cassette which is actually an ATP binding fatty acids throughout domain in the protein. They have played an important role to elucidate the the body. ABC mechanism of action of several drugs. For example, the ABC transporters of transporters are also bacterial plasma membrane are involved in transport of amino acid, sugar and present in simpler animals and in plants peptides. In eukaryotes, MDR protein is an important ABC transporter. Let us and microorganisms. first understand what is this ABC transporter and then we will focus on MDR protein. ABC transporters constitute a large family of ATP-dependent transporters that pump amino acids, peptides, proteins, metal ions, various lipids, bile salts, and many hydrophobic compounds including drugs, out of the cells against their concentration gradient. All ABC transporters have two ATP binding domains also known as nucleotide-binding domains (NBDs) and two transmembrane domains (Fig. 5.1 a). In some cases, all these domains are in a single long polypeptide chain; some ABC transporters have two subunits, each contributing an NBD and a domain with six (or in some cases twelve) transmembrane helices. Although many of the ABC transporters are in the plasma membrane, some types are also found in the intracellular organelles such as endoplasmic reticulum and in the membranes of mitochondria and lysosomes. Most ABC transporters act as pumps, but some members of the superfamily also act as ion channels that are opened and closed by ATP hydrolysis (Fig. 5.1.b). Some ABC transporters have very high specificity for a single substrate; others are more promiscuous. The CFTR transporter (cystic fibrosis transmembrane conductance regulator) is a chloride-channel regulated by ATP hydrolysis in the plasma membrane of epithelial cells and this too belongs to family of ABC transporters. 72 Unit 15 IntroducMtiemon btorane Biomem Transporbranet-sII ......

(a)

(b) Fig. 5.1: a) Pictorial representation of ABC transporters having two nucleotide- binding domains (NBDs) and two transmembrane domains (TMDs). b) Transport across ABC transporter. 5.2.1 Multidrug Resistance (MDR) protein

Multidrug Resistance (MDR) protein or P-glycoprotein because it includes a carbohydrate moiety is an ABC transporter in human beings. The multidrug The nucleotide binding transporter is responsible for the striking resistance of certain tumors to some domains of all ABC generally effective antitumor drugs. MDR has broad substrate specificity for proteins are similar in sequence and hydrophobic compounds, including, for example, the chemotherapeutic drugs presumably in three- adriamycin, doxorubicin, and vinblastine. By pumping these drugs out of the dimensional structure; cell, the transporter prevents their accumulation within tumor and thus nullifies they are the conserved their therapeutic effects. MDR is an integral membrane protein (MW 170 kd) molecular motor that can be coupled to a with 12 transmembrane segments and two ATP-binding domains wide variety of pumps (“cassettes”). and channels. The major cause of bacterial and cancer drug resistance is due to presence of MDR transporters that transports drug molecules out of the cell. MDR transporters can be divided into two classes based on their source of energy: secondary transporters, which use proton gradients to facilitate an antiport mechanism, and ABC transporters that couple the hydrolysis of ATP for transport of substrate across the cell membrane. In humans, 46 ABC transporters have been identified and play important roles in diseases, which include cystic fibrosis, macular dystrophy, and several neurological disorders. In most cases, these permeases have specific substrate specificities and may require a periplasmic binding protein to facilitate transport. The transmembrane helices, in contrast, are capable of recognizing and removing a large number of chemically unrelated lipids and 73 Block 12 MembBiraneomem Transporbranest ...... toxins directly from the cell membrane. Many of these transporters translocate useful cytotoxins such as anti-cancer drugs.

Tick [9] mark the correct option:

a) Molecular weight of MDR protein is 170 Da. [True/False]

b) ABC stands for ATP- binding codons. [True/False]

c) The CFTR transporter is a Na+ channel operated by ATP hydrolysis. [True/False] d) MDR transporters transport drug molecules out of the cell. [True/False]

5.3 GROUP TRANSLOCATION

Group translocation is another form of active transport common in prokaryotes. In this case a substance is chemically altered during its transport across a membrane so that once inside, the cytoplasmic membrane becomes impermeable to that substance and it remains within the cell. Now to understand group translocation, let us recall some facts. You know that the bonds between phosphate groups and other molecules are often referred to as high energy bonds. The breaking and reforming of such bonds involving phosphate is a powerful mechanism for transferring energy or using energy to drive cellular processes such as transportation across membranes. Also, many cellular processes involve phosphorylation, the transfer of phosphate groups from one molecule to another as a regulatory mechanism. In some cases phosphate is cleaved from a molecule such as ATP to form ADP and inorganic phosphate in a reaction that is coupled to another reaction that requires an input of energy. In other cases instead of energy, the transfer of a phosphate group may change the configuration of a molecule and affects its activity. A series of reactions may pass a phosphate group from molecule to molecule, performing cellular work, such as active transport, with each phosphorylation.

Group translocation is a process that can move solutes from a low to a high concentration, utilizing chemical energy in the form of phosphoenol pyruvate (PEP) and with the help of a number of proteins (cytoplamic and membrane). During transport the solute molecule is modified (phosphorylated by PEP) as it moves across membrane. The best studied group translocation system is the phospho-transferase system by which certain sugars are transported into cells. In prokaryotes, the phosphoenol pyruvate: carbohydrate phospho- transferase system (PEP: PTS) is the enzyme/transport system which phosphorylates its extracellular carbohydrate substrate as it is transported in the cell, resulting in the intracellular accumulation of the sugar phosphate esters. A number of phosphoryl transfer proteins are involved in sugar uptake especially glucose. PEP acts as the phosphoryl donor, and the incoming sugar as the ultimate phosphoryl acceptor. Fig. 5.2 depicts the difference between an 74 active transport and group translocation. Unit 15 IntroducMtiemon btorane Biomem Transporbranet-sII ......

Fig. 5.2: Difference between an active transport and group translocation.

The advantage of converting glucose into glucose-6-phosphate is that it does not affect the concentration gradient of glucose. Also, it will not leak out of the cell as the cell membrane is impermeable to glucose-6-phosphate.

Fill in the blanks with appropriate words: a) Group translocation is...... (active/passive) form of transport. b) During group translocation of glucose in the cell, glucose is phosphorylated to ...... (glucose 1, 6 bis phosphate/ glucose-6- phosphate). c) Chemical energy for group translocation of glucose is derived from ...... (Pyruvate/ PEP). d) In humans ...... (46/96) ABC transporters have been identified.

5.4 ION CHANNELS

Ion channels form a hydrophilic passage across the membrane through which water, specific ions or small hydrophilic molecules move down their concentration or electrical gradient across the biological membrane at a rapid The plasma membrane rate. Some of these channels which are open throughout are termed as non- of animal cells contain many open K+ channels gated channels while the ones which open in response to specific electrical but very few open Na+, or chemical signals are referred as gated channels. Gated channels are Ca2+ or Cl- channels. further classified into different types: voltage-gated ion channels, ligand- gated ion channels and mechano-gated channels. In this section you will learn in details about various types of these gated channels. 5.4.1 Voltage-gated ion channels

These ion channels are a class of transmembrane proteins that are activated by changes in membrane potential. One of their common features is the 75 Block 12 MembBiraneomem Transporbranest ...... existence of a voltage sensor domain in the channel structure. The voltage sensor is a region of the protein bearing charged amino acids that are The inside membrane potential (voltage) displaced by changes in the membrane electric field. Voltage-gated ion across the plasma channels are most abundant in the heart. They use charged membrane membrane of all cells is voltage sensor and auxiliary subunits that regulate channel protein trafficking negative to the range of and function. Fig. 5.3 gives the schematic representation of voltage-gated ion 50-70mV. channel.

Fig. 5.3: Schematic representation of voltage-gated ion channel.

Structurally voltage-gated ion channels possess similar molecular structure that includes a repeating motif with six membrane-spanning alpha helices. There is also a pore loop that contributes to the selectivity filter and a charged domain that acts as a voltage sensor. During depolarization, the inner face of the cell membrane becomes more positive and the voltage sensor (which Each of the homologous domains in a voltage carries a positive charge) is thrust upwards through the membrane by gated sodium channel electrostatic repulsion. This movement induces a conformational change in the has one transmembrane channel complex which opens the pore. Conductance changes steeply, domain in which a increasing 150-fold with a 10/ mV shift in membrane potential. Let us briefly positively charged amino acid is found at every look at the sodium and potassium channels as an example of voltage-gated third position, giving a ion channels. total of four to eight positive charges per The voltage-gated sodium channel is composed of a single large protein transmembrane domain. with four repeating motifs labelled I, II, III and IV (Fig. 5.4). Each motif contains the six membrane-spanning regions, a pore loop and a voltage sensor. In contrast, the voltage-gated potassium channel is composed of four separate subunits, each with the characteristic motif. The sodium channel is a specific example of a protein embedded in the plasma membrane. The subunit of the voltage-gated sodium channel is a polypeptide chain of more than 1800 amino acids with 24 transmembrane domains in the polypeptide chain. The channel has several functional parts. One portion of the channel determines its ion selectivity. This particular channel is quite selective for sodium ions. Even the chemically similar potassium ions cannot pass through the channel. Another portion of the channel serves as a gate that can open and close. The gate is controlled by a voltage sensor, which responds to the level of the membrane potential. The voltage sensor is represented as a transmembrane domain with fixed positive charges. At a typical resting membrane potential of -70mV the channel is closed. When any factor depolarizes the membrane potential sufficiently, the voltage sensor moves outward and the gates open.

76 Unit 15 IntroducMtiemon btorane Biomem Transporbranet-sII ......

Fig. 5.4: Schematic representation of voltage-gated sodium ion channel. 5.4.2 Ligand-gated ion channel

Unlike the voltage-gated ion channels which are activated by changes in membrane potential, ligand-gated ion channels are activated by some chemical signals/ molecules (Fig. 5.5). These ions channels are also known as ionotropic receptors. Binding of a ligand/ chemical messenger for example neurotransmitter to these transmembrane proteins allows the passage of ions such as Na+, K+, Ca2+ or Cl- ion. Let us go through some of the specific channels and their structural and functional significance.

Fig. 5.5: Schematic representation of ligand-gated ion channel.

One of the common ligand-gated ion channels is acetylcholine receptor (AChR) found in cell membranes. Binding of the neurotransmitter acetylcholine (Ach) to the receptor induces the opening of channel and thereby diffusion of sodium (Na+) and potassium (K+) ions through the pore occurs. These receptors can be further divided into two types, nicotinic and muscarinic. Nicotinic acetylcholine receptors (nAChR) are pentameric ligand-gated ion channels, whereas muscarinic acetylcholine receptors (mAChR) are seven- helix G-protein coupled membrane proteins. Now we will study the structural features of these receptors in detail. Nicotinic Acetylcholine Receptors (nAChR)

Nicotinic acetylcholine receptors or ligand-gated ion channels are found at the neuromuscular junctions. After binding of signal molecule to the receptors, they stimulate muscular contraction. They are composed of five subunits

(D2E3). The subunits are arranged to form a barrel shape creating a pore in the 77 Block 12 MembBiraneomem Transporbranest ...... centre (Fig. 5.6). The binding site for acetylcholine is on the two D subunits. The total protein complex starts from cytoplasm, spans through membrane and extends beyond the extracellular membrane. The binding of acetylcholine to D subunit induces a conformational change in the ion channel. This initiates the import of calcium and sodium ions and export of potassium ions from the cell.

(a) (b) Fig. 5.6: a) Membrane topology of a neuronal nAChR subunit. Each nAChR subunit contains four transmembrane domains (M1-M4) with a prominent M3-M4 intracellular loop of variable length. b) Five subunits co-assemble to form a functional subunit. Aquaporin abnormalities Muscarinic acetylcholine receptors (mAChR) cause various diseases in humans. Mutation Muscarinic acetylcholine receptors are G-protein receptors having seven leading to loss of transmembrane spanning regions. Different tissues have specific variation in function of AQP2 result in the disease these receptors. Five different types (M1-M5) have been reported. These nephrogenic diabetes receptors are involved in regulation of smooth muscle function, hormone insipidus, in which large secretion, heart rate and wakefulness. Neurotoxins inhibit the ion channel volume of dilute urine activity by reversible blocking acetylcholine binding site in the receptor. Due to are excreted. Mutations in AQP0 are associated blocking inhibiton, the formation of pore is prevented which results in inhibition with congenital of the transport of cations across the membrane. cataracts. The autoimmune disease 5.4.3 Mechano-gated channels neuromyelitis optica is caused by non-function Such channels are also referred as stretch-gated ion channels. They are of AQP4. useful for sense of touch-mediated response,hearing and balance. They are regulated by mechanical forces. Some examples are:

Aquaporins Aquaporins are family of small integral membrane proteins that are present throughout the animal and plant kingdom. The basic structure of aquaporin monomer consists of six transmembrane helical segments and two short helical segments. Cytoplasmic and extracellular sides are connected by a narrow aqueous pore. They contain several conserved motifs, including asparagine, proline, alanine (NPA, one alphabet notation for amino acids) sequences in their short helical segments. In membranes, aquaporin monomers assemble as tetramers with each monomer functioning independently (Fig. 5.7). The primary function of aquaporins is to transport water across cell membranes. Aquaporins play important role in fluid secretion and absorption across epithelial cells of kidney and exocrine glands. A subset 78 of aquaporins known as aquaglyceroporins also transports glycerol. Unit 15 IntroducMtiemon btorane Biomem Transporbranet-sII ......

Fig. 5.7: Structure of an aquaporin tetramer.

Aquaporins are also present in various plants and microbes, including bacteria and yeast. Plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins (TIPs) are major plant aquaporins. In plants, they are mainly involved in transpiration, metabolism and reproduction resulting in enhanced adaptation of plants to various environmental stresses. Bacteriorhodopsin

Bacteriorhodopsin (bR) is a seven-helical light-driven proton pump protein found in the purple membrane of the archea bacteria Halobacterium salinarium. It is light driven proton pump belonging to the family of archaeal rhodopsins, and exhibits a seven helical transmembrane architecture. It contains a buried retinal chromophore covalently bound to a conserved lysine residue in helix G via a protonated Schiff base. In Bacteriorhodopsin, number of charged residues and water molecules are arranged to form the proton translocation channel that extends across the membrane through the centre of the seven helical bundles. Light-induced structural changes cause these residues and water molecules to act as members of a relay team, passing a proton from the cytoplasmic to the extracellular side of the membrane. You will study more about bacteriorhodopsin in Unit 12 of Block 4.

Fill in the blanks: a) The subunit of voltage gated sodium channel is a polypeptide of about ...... (1800/180) amino acids.

b) Nicotinic acetylcholine receptor is composed of ...... (5/8) subunits. c) Aquaporin monomer consists of ...... (four/six) transmembrane helical segments. d) Bacteriorhodopsin is a seven-helical light-driven ...... (sodium/ proton) pump. 79 Block 12 MembBiraneomem Transporbranest ......

Tick [9] mark the correct option:

a) Bacteriorhodopsin is a seven helical light driven proton pump. [True/False] b) Muscaranic acetylcholine receptors are G-protein coupled receptors. [True/False]

c) Bacteriorhodopsin contains a buried retinal chromophore covalently bound to a conserved lysine residue. [True/False]

d) Aquaporins in plants are mainly involved in transpiration and in enhanced adaptation to various environmental stresses. [True/False]

5.5 IONOPHORES

Ionophores are low molecular weight small molecules that facilitate transport of ions across the cell membranes. They bind to particular ions and act as a mobile carrier, transporting them through the hydrophobic membranes. In some cases they form ion channels which create pores in membranes through which ions can move. They are of two types, carrier ionophores (mobile type) or channel forming ionophores (fixed type). For instance, various microorganisms produce several toxin molecules which enhance the transport of ions across the membranes such as valinomycin, gramicidin, ionomycin etc. They are quite simple molecules and act as mobile carriers or pores in membrane transport. These mobile carriers bind to specific ions and diffuse across the membrane. Pores create a hole in the membrane, which allows the movement of ions. Structurally, the pore may be a monomeric or multimeric protein embedded in the membrane. 5.5.1 Valinomycin

An ionophore, valinomycin is an example of carrier ionophore that is antibiotic molecule secreted by bacterium Streptomyces which also acts as a carrier for potassium ion. It consists of 12 units of alternating amino acids and esters. The aminoacids that comprise valinomycin are d- and l-valine (hence the name “valinomycin”), hydroxyvaleric acid and l-lactic acid. Major effect of valinomycin is on energy dependent swelling of mitochondria, release of cytochrome C, oxidation of NADPH and apoptosis. Valinomycin is also used for diagnostic purpose in some diseases because it is highly specific for K+ ions. The ability to transport Na+ is at least 104 times less than that for K+. 5.5.2 Gramicidin

Gramicidin is example of channel forming ionophore that is mixture of antibiotic peptides obtained from the soil bacterium Bacillus brevis. It also works as an ion channel. It is a linear peptide of 15 amino acids having d- and l- amino acids. In membranes, dimerization of gramicidin leads to its folding as a right handed E-helix spanning the lipid bilayer and makes the membrane permeable to protons as well as alkali metal ions. Abundance of hydrophobic residues in the Gramicidin molecule results in its rapid incorporation into lipid 80 bilayer and other membranes and stimulates the diffusion of many cations. A Unit 15 IntroducMtiemon btorane Biomem Transporbranet-sII ...... single gramicidin channel is able to transport more than 10 million K+ ions per second. The divalent cations such as Ca2+cannot pass through the channel and thus block the channel. Fig. 5.8 pictorially describes the functioning of gramicidin. When the two gramicidin molecules join end to end, they span the membrane and form a channel for ion to pass through.

Gate Open Gate Closed

Fig. 5.8: Dimerization mechanism of Gramicidin gating.

Fill in the blanks: a) Ionophores are...... (high/low) molecular weight small molecules that facilitate transport. b) Gramicidin consists of ...... (15/25) amino acids.

c) Valinomycin is a carrier for ...... (K+/Mg2+).

d) Dimerization of gramicidin molecules lead to ...... (opening/closing) of gates.

5.6 SUMMARY

Let us recapitulate what we have learnt so far:

x ABC proteins are important transport proteins found in all organisms ranging from bacteria to mammals. These ATP dependent pumps are also known as ATPase. ABC transporters constitute a large family of ATP- dependent transporters that pump amino acids, peptides, proteins, metal ions, various lipids, bile salts, and many hydrophobic compounds including drugs, out of the cells against their concentration gradient.

x All ABC transporters have two ATP binding domains also known as nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs).

x Multidrug resistance (MDR) protein is an important ABC transporter. MDR protein pumps hydrophobic drugs from cytosol to outside of the cell. These transporters play important role in removing harmful substances (like drugs and other toxins) from the cells.

x Group translocation is a process in which solutes are transferred from their low concentration to high concentration, utilizing chemical energy derived from phosphorylation especially by phosphoenol pyruvate (PEP). 81 Block 12 MembBiraneomem Transporbranest ...... x Some ion channels may remain open throughout and are termed as nongated channels while the ones which open in response to specific electrical or chemical signals are referred as gated channels. Gated channels may respond to change in membrane potential and are known as voltage-gated ion channels. Ligand-gated ion channels bind to specific molecules for activation.

x The acetylcholine receptor (AChR) is a ligand-gated ion channel that binds to the neurotransmitter acetylcholine.

x Aquaporins are family of small integral membrane proteins that are present throughout the animal and plant kingdom.

x Bacteriorhodopsin is a seven-helical light-driven proton pump.

x Valinomycin is an antibiotic which acts as a potassium (K+) ionophore by inducing K+ conductivity in cell membranes. 5.7 TERMINAL QUESTIONS

1. Describe the structure of ABC transporters. 2. Discuss the mode of action of MDR transporters. 3. Explain the steps of group translocation of glucose in the cell.

4. Write a note on voltage gated sodium channel.

5. Explain the functions of acetylcholine receptors. 6. What are aquaporins? Describe their importance. 7. What are ionophores? 5.8 ANSWERS Self-Assessment Questions

1. a) true b) false c) false d) true 2. a) active b) glucose-6- phosphate c) PEP d) 46

3. a) 1800 b) five c) four d) proton 4. a) true b) true c) true d) true

5. a) low b) 15 c) K+ d) opening Terminal Questions

1. ABC transporters have two nucleotide-binding domains (NBDs) and two transmembrane domains. In some cases, all these domains are in a single long polypeptide; other ABC transporters have two subunits, each contributing an NBD and a domain with six (or in some cases ten) transmembrane helices. For details refer section 5.2. 2. The multidrug transporter (MDR) is responsible for the striking resistance of certain tumors to some generally effective antitumor drugs. MDR has a broad substrate specificity for hydrophobic compounds, including, for 82 Unit 15 IntroducMtiemon btorane Biomem Transporbranet-sII ...... example, the chemotherapeutic drugs adriamycin, doxorubicin, and vinblastine. By pumping these drugs out of the cell, the transporter prevents their accumulation within a tumor and thus nullifies their therapeutic effects. For details refer section 5.2.

3. Group translocation is a process that can move solutes from low to a high concentration, utilizing chemical energy in the form of phosphoenol pyruvate (PEP). This process requires a number of cytoplamic and membrane proteins, and the solute molecule is modified (phosphorylated by PEP) as it moves across membrane. For details refer section 5.3

4. The sodium channel is a specific example of a protein embedded in the plasma membrane. The subunit of the voltage-gated sodium channel is a polypeptide chain of more than 1800 amino acids with 24 transmembrane domains in the polypeptide chain. The voltage-gated sodium channel is composed of a single large protein with four repeating motifs labelled I, II, III and IV. Each motif contains the six membrane-spanning regions, a pore loop and a voltage sensor. One portion of the channel determines its ion selectivity. This particular channel is quite selective for sodium ions. Even the chemically similar potassium ions cannot pass through the channel. For more details refer section 5.4.1.

5. Acetylcholine receptors (AChR) bind to the neurotransmitter acetylcholine (Ach) which induces the opening of channel and thereby diffusion of sodium (Na+) and potassium (K+) ions through the pore occurs. For more details refer section 5.4.2. 6. Aquaporins are family of small integral membrane proteins that are present throughout the animal and plant kingdom. The primary function of aquaporins is to transport water across cell membranes. Aquaporins play important role in fluid secretion and absorption across epithelial cells of kidney and exocrine glands. A subset of aquaporins known as aquaglyceroporins also transports glycerol. In plants, they are mainly involved in transpiration, metabolism and reproduction resulting in enhanced adaptation to various environmental stresses. 7. Ionophores are low molecular weight small molecules that facilitate transport of ions across the cell membranes. They bind to particular ions and act as a mobile carrier, transporting them through the hydrophobic membranes. In some cases they form ion channels which create pores in membranes through which ions can move. For example several toxin molecules such as valinomycin, gramicidin, ionomycin etc produced by microorganisms enhance the transport of ions across the membranes and are known ionophores. For more details refer section 5.5. 5.8 SUGGSETED READINGS

x Garret, R.H., Grisham, C.M. (2016). Biochemistry (6th ed.). Boston, Cengage Learning. ISBN-10: 1133106293, ISBN-13: 978-1133106296

x Berg, J.M., Tymoczko, J.L. and Stryer L., (2012) W.H. Biochemistry (7th ed.), Freeman and Company (New York), ISBN:10: 1-4292-2936-5, ISBN:13:978-1-4292-2936-4. 83 Block 12 MembBiraneomem Transporbranest ...... x Nelson, D.L., Cox, M.M. (2017). Lehninger: Principles of Biochemistry (7th ed.). New York, WH: Freeman and Company. ISBN: 13: 978-1-4641-2611-6 / ISBN:10:1-6412611-9.

x Voet, D.J., Voet, J.G., Pratt, C.W. (2008). Principles of Biochemistry (3rd ed.). New York, John Wiley & Sons, Inc. ISBN:13: 978-0470-23396-2

84 Unit 1 Introduction to Biomembranes ......

UNIT 6

VESICULAR TRANSPORT AND MEMBRANE FUSION

Structure 6.1 Introduction 6.4 Molecular Mechanism of Vesicular Transport Expected Learning Outcomes Membrane Fusion 6.2 Vesicular Transport: Types and Functions Receptor Mediated Endocytosis Cisternal Maturation Model 6.5 Summary Vesicular Transport Model 6.6 Terminal Questions 6.3 Transport Vesicles 6.7 Answers Clathrin-Coated Vesicles 6.8 Suggested Readings COP-Coated Vesicles (COPI and COPII)

6.1 INTRODUCTION

In the previous units you learnt about various types of membrane transport and transporters. You also studied their role in maintaining the life processes at cellular level. Membrane transport proteins and various carrier molecules present in the membrane allow the movement of ions or molecules through semipermeable or selectively permeable biological membrane even at the cost of energy. In this unit you will learn about vesicular transport that is essential for the transportation of soluble and membrane proteins to their destinations. We will also focus on the various types of vesicular transport vesicles and molecular mechanism of vesicular transport. We will also deal with receptor mediated endocytosis in section 6.5 about which you have studied briefly in Unit 4. 85 Block 12 MembBiraneomem Transporbranest ...... Expected Learning Outcomes After studying this unit, you should be able to:

write the significance of vesicular transport;

differentiate between clathrin and COP coated vesicles;

describe the molecular mechanism of vesicular transport;

explain membrane fusion; and

describe receptor mediated endocytosis. 6.2 VESICULAR TRANSPORT

You may revisit about protein from Cell biology (BBCCTT-103) course that you studied in first semester (unit-9). The transport across plasma membrane and vesicular transport are two different phenomena. During membrane transport the transfer of ions or molecules takes place across the membrane while vesicular transport deals with the soluble and membrane proteins i.e. proteins destined along the secretory route. The synthesis and their translocation between different organelles of cell are facilitated by small membrane bound structures known as transport vesicles. In eukaryotes, vesicular transport is crucial for growth, development and survival of organism because it maintains the correct balance and distribution of various moieties in specific cellular compartments. The selectivity of such transport is important for maintaining the functional organization of the cell. Thus, this transport process must be precise and operate in optimal manner. This is possible due to the presence of specific receptor proteins and signaling molecules present on both the vesicles and target sites. Recall the co-translational translocation of proteins you have studied in Cell Biology (BBCCT-103 Block III: Protein Trafficking). Proteins destined along the secretory route are transported from endoplasmic reticulum (ER) to Golgi complex for post translational modification as well as sorting. The proteins are transported to lysosomes, secretory granules or plasma membrane according to the signal it bears which is encoded by their sequence. Vesicular transport mechanism helps in sorting and transport of protein to their correct address. A simple question comes to mind. How do these proteins maintain their integrity? To answer this question let us go through vesicular transport models. The Golgi has been classified into: 1. cis compartment closest to ER 2. medial compartment 3. trans compartment, which exports proteins to different destinations.

If you remember the co-translational modification of proteins, the core glycosylation takes place in ER. In each compartment of Golgi, the carbohydrate units are specifically added or modified. In cis Golgi three mannoses are removed from the oligosaccharide chain of proteins to be secreted or to be inserted in the plasma membrane. In medial Golgi, two 86 Unit 16Vesicular TransporIntroduct antiond Mtoem Bibomemrane Fbusraneions ...... more mannoses are removed and two N-acetylglucosamines and a fucose is added. Finally, in trans Golgi, another N-acetylglucosamine is added followed by addition of galactose and sialic acid. Fig. 6.1shows the path how Golgi apparatus modifies and sorts proteins for transport throughout the cell as well as helps in maintaining membrane integrity.

What happens when there are defects in Golgi function?

Fig. 6.1: Protein modification and sorting in Golgi apparatus for transport Defects in various throughout the cell. aspects of Golgi function can result in In vesicular transport, membrane symmetry is preserved i.e. the cytosolic congenital glycosylation face of a transport vesicle corresponds to the cytosolic face of donor disorders, some forms of muscular dystrophy, compartment. After fusion, the cytosolic face of the transport vesicles and may contribute to becomes continuous with the cytosolic face of the target compartment. diabetes, cancer, and Accordingly, when a vesicle fuses with the plasma membrane, its luminal cystic fibrosis. surface becomes part of the external side of the plasma membrane. The carbohydrate groups of glycoproteins in plasma membrane are always on the extracellular surface. There are two opposing models to explain the movement of proteins through Golgi: Cisternal maturation model and Vesicular transport model. 6.2.1 Cisternal Maturation Model

How do cargo proteins move between the Golgi cisternae? As it was mentioned earlier, Golgi-mediated modifications act as signals to direct the proteins to their final destinations within cells, including the lysosome and the plasma membrane. Initially scientists believed that each Golgi cisterna was transient and that the cisternae themselves moved from the cis to the trans face of the Golgi, changing over time. So, proteins were travelling as passengers (cargo) within cisternae. Cisternal maturation model proposes 87 Block 12 MembBiraneomem Transporbranest ...... that the enzymes present in each individual cisterna change over time, while the cargo proteins remain inside the cisterna. However, another school of researches believe in the vesicular transport model. 6.2.2 Vesicular Transport Model

It is believed by a group of scientists that instead of entire cisternae, vesicles formed in the Golgi move cargo proteins. This model was given by George Palade and Marilyn Farquhar in 1998. The vesicular transport model proposes that the Golgi cisternae are a stable compartment which carries protein modification enzymes (such as enzymes to add or remove sugars, add sulfate groups, and perform other modifications). Vesicles arrive at each cisternae carrying cargo proteins; and there it undergoes modification by the resident enzyme. New vesicles carrying the modified protein buds off from the cisternae, and travel to the next stable cisternae till the modification is complete.

To summarize, one model says that cisternae are transient structures and each cisternae physically moves from cis to trans face while the modification goes on. Another model is of the view that cisternae are stable structures having specific enzymes. And vesicles carrying cargo proteins move from one cisternae to another for modification to take place. It is yet debatable which model is best. Now, the question arises, Do all proteins follow the same path?

Tick [9] mark the correct option: a) Cis Golgi compartment is closest to ER. [True/False] b) Post translational modification occurs in lysosomes. [True/False] c) During glycoprotein synthesis, fucose unit is attached in ER. [True/False] d) Specific receptor proteins are present only on the receptors and not on the vesicles. [True/False]

e) The carbohydrate group of glycoproteins in plasma membrane is always present on the extracellular side. [True/False] f) Vesicular transport model proposes that the enzymes present in each individual cisterna change over time, while the cargo proteins remain inside the cisterna. [True/False]

Answer in 1-2 sentences: a) Define vesicular transport...... b) How transport across membrane is different from vesicular transport? ...... 88 Unit 16Vesicular TransporIntroduct antiond Mtoem Bibomemrane Fbusraneions ...... c) Name the models of vesicular transport mechanism......

6.3 TRANSPORT VESICLES

As you already know function of coated vesicles is related to endocytosis and the intracellular transport of membranes and soluble proteins. These coated vesicles vary in size between 50 to 250 nm and are characterised by the presence of a coat made of tiny, regularly spaced bristles that cover the cytoplasmic side of the vesicle. A vesicle is formed by budding off from a donor membrane that in turn fuses with an acceptor membrane. Sometimes donor membrane undergoes distortion to initiate budding. The first step in vesicular transport is the formation of a coated vesicle by budding from the donor membrane surface. Coated vesicles are involved in the vesicular transport throughout the endomembrane system, as well as in exocytosis and endocytosis. The nature of coat depends on the cargo protein to be transported and the targeted destination/ acceptor membrane. Here, you must note that the protein coat of the coated vesicle is formed from soluble proteins on the cytosolic face of the donor membrane guided by G-proteins. The involvement of GTPases like dynamin has been shown in the formation of vesicles. It forms a ring around the neck of the bud and GTP hydrolysis constricts the ring, pinching off the neck to release the vesicle (Fig. 6.2). Transport of vesicles occurs with the help of cytoskeletal proteins such as actin filaments to the targeted site. Let us focus on some of the best characterised coated vesicles.

1. Clathrin-Coated Vesicles 2. COP-Coated Vesicles (COP I and COP II)

Fig. 6.2: Main steps of the clathrin coated vesicle formation. 89 Block 12 MembBiraneomem Transporbranest ...... 6.3.1 Clathrin-Coated Vesicles

Clathrin-coated vesicles are one of the most extensively characterized transport vesicles. They mediate endocytosis of transmembrane receptors and transport of newly synthesized lysosomal hydrolases from the trans-Golgi network to the lysosome. Accumulation of clathrin on the plasma membrane stimulates the formation of clathrin coated vesicles. The cell surface receptors (adaptor protein complexes), interact with each other, with membranes, and with the sorting signals found on cargo molecules. Coat constituents not only serve to shape the budding vesicle, but also play a direct role in the packaging of cargo, suggesting that protein sorting and vesicle budding are functionally integrated.

One clathrin molecule is comprised of three molecules of heavy-chain and three molecules of light chain and is called a triskelion. Each heavy chain of the structure is of 180 kDa whereas each light chain has the molecular weight of 35 kDa. Clathrin molecules successively assemble into a polyhedral, cage- like coat on the surface of the coated pit. Triskelion, a three-pronged protein complex made up of clathrin coat attaches to the membrane via an adaptor protein (AP) complex. Adaptor proteins bind both to clathrin and to integral membrane proteins of the vesicle and stimulate its assembly. Much more importantly, by binding to the molecules in the membrane of the vesicle, adaptor proteins appear to be responsible for recognising the appropriate cargo molecules.

Main steps of the clathrin coated vesicle formation are shown in (Fig. 6.2):

1. Recruitment of the G-protein, adaptor proteins and clathrin to defined sites on the plasma membrane.

2. Clathrin concentrates in specific areas of the plasma membrane, forming clathrin-coated membrane invaginations, called clathrin-coated pits.

3. Budding and detachment of the nascent clathrin-coated vesicles through a series of highly regulated steps.

6.3.2 COP-Coated Vesicles (COP I and COP II)

COP-coated vesicles (COP I and COP II) transport all type of molecules from the Golgi to the ER and back. Various steps of vesicular transport is assisted by specific type of coat structures. For transport of molecules from cis Golgi to ER, COP I coated vesicles are involved, whereas in reverse transport i.e. from ER to Golgi, COP II coated vesicles are used. This differential selectivity is helpful in creation of one way transport system of specific molecules to corresponding organelles in the cell. By this route, the newly synthesized proteins are targeted to specific locations in the cell or secreted out of the cell. The ER-resident proteins have ER retention signals at the c-terminal. The KDEL(Lys-Asp-Glu-Leu) for lumen protein and KKxx(Lys-Lys-?-?) for membrane protein. In Table 6.1 details of sorting signals that direct proteins to specific transport vesicles is given.

90 Unit 16Vesicular TransporIntroduct antiond Mtoem Bibomemrane Fbusraneions ...... Table 6.1: Sorting signals that direct proteins to specific transport vesicles

Signal Sequence Proteins with Signal Receptor Type of Transport Signal Vesicles

Luminal Sorting Signals

Lys-Asp-Glu-Leu ER-resident KDEL receptor COP I (KDEL) soluble proteins

Cytoplasmic Sorting Cells

Lys-Lys-X-X (KKXX) ER-resident COP I D and COP I membrane Esubunits proteins

Di-acidic (e.g., Asp- Cargo COP II Sec 24 COP II X-Glu) membrane subunit proteins

Asn-Pro-X-Tyr LDL receptor AP2 complex Clathrin/AP2

Tyr-X-X-I(YXX I) Membrane AP1 and AP2 Clathrin/AP1 and proteins AP2

Leu-Leu (LL) Plasma AP2 complexes Clathrin/AP2 membrane proteins

The molecules to be transported through COP I-coated vesicles are selected by binding of the molecules to specific membrane receptors. Transport by COP I-coated vesicles pathway is often called the retrograde transport pathway (Fig. 6.3) because it is involved in the ER retrieval pathway. COP II coated vesicles are required for transport of molecules from ER to Golgi. This mode of transportation is also known as anterograde transport pathway.

Fig. 6.3: Various types of vesicular transport pathways.

COP I coated vesicles are formed by sequence of events much more similar to that for Clathrin coated vesicles. In case of COP II coated vesicles, the final structure of coat is formed by sequential assembly of specific protein 91 Block 12 MembBiraneomem Transporbranest ...... components. Fig. 6.4 gives an overview of vesicle formation of both the types.

Fig. 6.4: Formation of Clathrin-coated vesicles involved in the endosomal pathways; COPI-coated vesicles involved in retrograde transport from the trans Golgi back through the Golgi cisternae to the ER and COPII-coated vesicles for transport from the ER to the cis Golgi. G protein Sar1 is responsible for initiation of budding of COP II coated vesicles. Additionally, two other proteins Sec 23 and Sec 24 are part of the pre-budding complex. The mechanism of action and formation of pre-budding complex is quite different for clathrin and COP II coated vesicles.

Fill in the blanks with appropriate words: a) The coat on vesicle for transport of cargo from ER to Golgi is made up of ……… (G-protein/ COP II). b) Budding of COP II vesicles is initiated by G protein …… (Sar 1/ SNARE).

c) Clathrin-coated vesicles mediate …………… (exocytosis/ endocytosis) of transmembrane receptors and transport of newly synthesized lysosomal hydrolases from the trans-Golgi. d) ………. (ATPases/ GTPases) are involved in the formation of vesicles.

6.4 VESICULAR TRANSPORT MECHANISM

In the previous section we discussed about different types of vesicle formation. In this section we will learn how these vesicles are transported from their site 92 of formation to their designated targets resulting in membrane fusion. Unit 16Vesicular TransporIntroduct antiond Mtoem Bibomemrane Fbusraneions ...... The vesicular transport is initiated after the formation of the transport vesicles. Vesicles are formed by detachment of a small portion of lipid bilayer. This process is also called budding. The initiation of membrane curvature required for vesicle formation requires energy. This process is mediated by proteins such as epsins. The newly formed vesicles contain the proteins that were present in the portion of membrane as well as soluble molecules. Fusion of the vesicle with a target membrane is generally a reversal of the process of its formation. The proteins that mediate targeting of the vesicle to the specific cellular location also mediate fusion, and in some systems regulate the precise time at which fusion occurs.

The molecular components of vesicular transport have been discovered using mutant strains of yeast. The formation of transport vesicles and selective Hsc70 is a chaperone incorporation of cargo into them is mediated by their protein coats. The coats protein with diverse are supramolecular assemblies of protein on nascent vesicles. The coat cellulose functions also formation initiates the shaping of curvature on the flat membranes. The coat known as HSPA8. proteins are also involved in the recognition of sorting signals in the cargo. ATPase activity of Hsc70 is important for Most of the cargo proteins are found in COP II coated buds and vesicles while endocytosis during the uncoating of clathrin being exported from ER. These proteins may be attached directly to COP II or coated resides. are indirectly bound to COP II through some transmembrane export receptors. The sorting signals recognized by COP II are present in the cytosolic domains of transmembrane cargo proteins. Structurally clathrin coats are more complex than COP II and COP I. Clathrin vesicle assembly is regulated by kinases, phosphatases and other accessory proteins. Scission of clathrin coated vesicles depends on accessory factors such as, dynamin. In addition, the uncoating of clathrin vesicle is mediated by the chaperons Hsc 70 and auxilin.

After uncoating, the vesicles are targeted to corresponding acceptor NEM- compartment where it is fused with the acceptor membrane. N- n-ethylmaleimide ethylmaleimide sensitive factor (NSF) binds to D- SNAP (soluble NSF NSF – attachment protein) to form a complex molecule. This complex binds to n-ethylmaleimide (NEM) Sensitive Factor membrane associated receptors called SNAREs. Each type of transport vesicle contains a specific v-SNARE that binds to corresponding t-SNARE SNAP- on the target membrane. Most SNAREs have C- terminal anchored Soluble NSF Attachment Proteins transmembrane proteins while their N-terminal domain faces the cytosol. They contain a heptad (group or set of seven) repeat motif of 60-70 amino acids SNAREs (v- and t-)- Soluble NSF Attachment which participate in coiled coil formation. The SNARE complex formed by the protein Receptors pairing of v-SNARE and t-SNARE is very stable four helix bundle in which v- SNARE contribute one helix and t-SNARE the remaining three D-helices.

SNAREs seem to perform two major functions.

i) Fusion of membrane

ii) Provide specificity of membrane fusion

Fig. 6.5 gives an overview of various steps of vesicle budding and fusion. As you can note from the figure. Specificity is also provided by tethering proteins that assist in membrane fusion prior to SNARE complex formation. 93 Block 12 MembBiraneomem Transporbranest ......

Fig. 6.5: Various steps of vesicle budding and fusion. 6.4.1 Membrane Fusion

Membrane fusion during vesicle transport consists of two reactions. First, the vesicles have to select and recognize the specific target membrane according to its receptor signal. After this, the two membranes fuse and deliver the content of vesicles to the targeted cellular compartment. The specificity is provided by a group of proteins called SNAREs. As you have read in the previous section SNAREs are of two types: v-SNARE and t-SNARE; v- SNAREs are found on vesicles whereas t-SNARES are present on the target membrane. The initial step is membrane recognition followed by a loose interaction called tethering. In the later stages, the membranes reside much closer to each other, known as docking which ultimately results in membrane fusion. In addition to SNAREs, two other types of proteins are also required for vesicle membrane fusion. The Rab proteins are a family of small GTP-binding proteins that are related to the Ras proteins. After the formation of complexes between complementary SNAREs and membrane fusion, a complex of two additional proteins (the NSF/ SNAP complex) is needed to complete the process of vesicle transport. These are not required directly for either vesicle/ target pairing or for the fusion of paired membranes. However, the NSF/SNAP proteins act after membrane fusion to disassemble the SNARE complex and thus the components of SNAREs are reutilized for other rounds of vesicle transport. Look at Fig.6.6 to visualize the steps involved in interaction and fusion of the membranes of the vesicle and the target and recycling of SNAREs. The interaction and fusion of the membranes of the vesicle and the target involves following steps:

1. Specification of the vesicle delivery site,

2. Recruitment of components capable of initiating vesicle ‘capture’, 3. Formation of a bridge between the vesicle and the target membrane, 94 Unit 16Vesicular TransporIntroduct antiond Mtoem Bibomemrane Fbusraneions ...... 4. Conformational change that allows the vesicle and target membrane proteins to come close enough to interact,

5. Dissociation of the tethering proteins, to free them for another round of transport.

The tethering process is regulated by a multimeric protein complex, called TRAPPs (Transport Protein Particles). It is made of 10 subunits and has the molecular weight of about 1,100 kDa. All the proteins of TRAPP complex are highly conserved and are found embedded in the membrane.

Fig. 6.6: Steps involved in interaction and fusion of the membranes of the vesicle and the target and recycling of SNAREs.

Fill in the blanks: a) Uncoating of clathrin vesicle is mediated by ………. (NSF/ chaperons).

b) Vesicle fusion is mediated by interactions between specific pairs of protein called ………… (SNAREs/ COP II).

c) Transport protein particle (TRAPP) complex is made of …….. (two/ ten) subunits. d) v-SNAREs are found on ……… (target membrane/ vesicles) whereas t-SNARES are present on the (target membrane/ vesicles).

95 Block 12 MembBiraneomem Transporbranest ...... 6.4.2 Receptor Mediated Endocytosis Some proteins enter the cell from the surrounding medium through the process of receptor-mediated endocytosis. The receptor-mediated endocytosis is a process for import of specific materials in the cell. A specific structure called endosome is formed. The receptors on the surface of endosomes are specific and selective. The receptors found on the cell membrane bind to the corresponding ligand/ molecule before its transfer in the cell. The process has following steps: 1. Exogenous ligand binds to specific membrane receptors

2. Formation of clathrin coated vesicles

3. Membrane invagination

4. Vesicle uncoating

5. Compartment of uncoupling receptors and ligand (CURL) and endosome formation

6. Ligand is further modified by the cell, then 7. Receptors are recycled towards the surface or digestive vacuoles, and 8. Receptors merges with cell membrane. In eukaryotes, the clathrin-mediated endocytic pathway is involved in the selective intake of proteins at the plasma membrane. The clathrin deposition on the membrane initiates the vesicle formation and process of endocytocis of molecules or ligands from outside of the cell to its interior. After the clathrin molecules are assembled, the membrane is deformed to form the vesicle by making an inward curvature. Several ligands can be imported in the cell by a single coated pit. After this process is complete, the clathrin coat is lost and its components are recycled. The transport of cholesterol through low density lipoprotein (LDL) is mediated by receptor mediated endocytosis. This is a crucial process through which cholesterol is distributed to all the cells. When LDL receptors become non-functional, the cholesterol is not released into the cell and LDL remains in the blood stream thereby causing problems to the patients. 6.5 SUMMARY

Let us recapitulate what we have learnt so far:

x Proteins are transported from endoplasmic reticulum (ER) to Golgi complex for post translational modification (addition and alteration) as well as sorting.

x Vesicle formation requires deformation of the lipid bilayer, forming a goblet- shaped invagination of the membrane that will eventually be pinched off to form the vesicle, a process called budding.

x COP I-coated vesicles shuttle molecules from exit sites on the cis Golgi complex towards the ER, while COP II-coated vesicles shuttle them from the ER towards the Golgi. 96 Unit 16Vesicular TransporIntroduct antiond Mtoem Bibomemrane Fbusraneions ...... x Most of the cargo proteins are found in COP-II coated buds and vesicles while being exported from ER.

x Each type of transport vesicle contains a specific v-SNARE that binds to corresponding t-SNARE on the target membrane.

x Receptor-mediated endocytosis is an extremely selective process of importing materials into the cell. This specificity is provided by receptor proteins located on specific areas of the cell membrane and is known as coated pits. 6.6 TERMINAL QUESTIONS

1. Name the different compartments of Golgi and associated functions. 2. Differentiate between anterograde and retrograde transport pathway. 3. Write down the steps of clathrin coated vesicle formation. 4. Explain the role of SNAREs in vesicular transport. 5. Explain membrane fusion. 6. Discuss the events involved in target specificity. 7. Enlist various steps of receptor mediated endocytosis. 6.7 ANSWERS

Self-Assessment Questions

1. a) true b) false c) false d) false e) true f) false 2. a ) Vesicular transport is a mechanism which helps in sorting and transport of protein to their correct address such as lysosomes, secretory granules or plasma membrane according to the signal it bears. b) Transport across plasma membrane and vesicular transport are two different phenomena. During membrane transport the transfer of ions/ molecules takes place across the membrane while vesicular transport deals with the soluble and membrane proteins i.e. proteins destined along the secretory route.

c) Cisternal maturation model and vesicular transport model. 3. a) COP-II b) Sar 1 c) endocytosis d) GTPases 4. a) chaperons b) SNAREs c) ten d) target membrane Terminal Questions

1. The different compartments of Golgi are:

i) cis compartment closest to ER ii) medial compartment iii) trans compartment, which exports proteins to different destinations. For details refer section 6.2. 97 Block 12 MembBiraneomem Transporbranest ...... 2. Transport of secretory molecules from ER to Golgi is known as anterograde transport pathway and COP II coated vesicles are involved in this transportation whereas transport by COP I-coated vesicles pathway is often called the retrograde transport pathway. It is also involved in the ER retrieval pathway. Refer section 6.3.2.

3. Vesicle formation requires deformation of the lipid bilayer, forming a goblet- shaped invagination of the membrane that will eventually be pinched off to form the vesicle, a process called budding. Three vesicle formation are

i) Recruitment of the G-protein, adaptor proteins and clathrin at the defined sites on the plasma membrane,

ii) Clathrin concentrates in specific areas of the plasma membrane, forming clathrin-coated membrane invaginations, called clathrin- coated pits.

iii) Budding and detachment of the nascent clathrin-coated vesicles through a series of highly regulated steps. Refer section 6.3.1. 4. SNAREs are membrane associated receptors and play a very important role in vesicular transport. They provide specificity in transportation. Each type of transport vesicle contains a specific v-SNARE that binds to corresponding t-SNARE on the target membrane. Refer section 6.4. 5. The interaction and fusion of the membranes of the transport vesicle with its target involves two types of events. First, the transport vesicle must specifically recognize the correct target membrane; for example, a vesicle carrying lysosomal enzymes has to deliver its cargo only to lysosomes. Second, the vesicle and target membranes must fuse; thereby delivering the contents of the vesicle to the target organelle. Refer section 6.4.1. 6. SNAREs play an important role in determining target specificity. Vesicle fusion is mediated by interactions between specific pairs of proteins, called SNAREs, on the vesicle and target membranes (v-SNAREs and t- SNAREs, respectively). Membrane fusion as a series of events includes: i) Specification of the vesicle delivery site

ii) Recruitment of components capable of initiating vesicle ‘capture’ iii) Formation of a bridge between the vesicle and the target membrane iv) Conformational change that allows the vesicle and target membrane proteins to come close enough to interact

v) Dissociation of the tethering proteins, to free them for another round of transport. Refer section 6.4 for more details. 7. Receptor mediated endocytosis consists of following steps:

i) Exogenous ligand binds to specific membrane receptors. ii) Clathrin vesicles are formed. iii) Membrane invagination iv) Vesicle uncoating 98 Unit 16Vesicular TransporIntroduct antiond Mtoem Bibomemrane Fbusraneions ...... v) Compartment of uncoupling receptors and ligand (CURL) endosome forms.

vi) Ligand is further modified by the cell

vii) Receptors are recycled towards the surface or digestive vacuoles

viii) Receptors merge with cell membrane. Refer Section 6.4.2. 6.8 SUGGESTED READINGS

x Garret, R.H., Grisham, C.M. (2016). Biochemistry (6th ed.). Boston, Cengage Learning. ISBN-10: 1133106293, ISBN-13: 978-1133106296 x Berg, J.M., Tymoczko, J.L. and Stryer L., (2012) W.H. Biochemistry (7th ed.), Freeman and Company (New York), ISBN:10: 1-4292-2936-5, ISBN:13:978-1-4292-2936-4.

x Nelson, D.L., Cox, M.M. (2017). Lehninger: Principles of Biochemistry (7th ed.). New York, WH: Freeman and Company. ISBN: 13: 978-1-4641-2611- 6 / ISBN:10:1-6412611-9.

x Voet, D.J., Voet, J.G., Pratt, C.W. (2008). Principles of Biochemistry (3rd ed.). New York, John Wiley & Sons, Inc. ISBN:13: 978-0470-23396-2