Molecular Biology of the Cell - Volume 1 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

Molecular Organization of Cells
The Plasma Membrane
Membrane Proteins

Although the basic Structural Features of Introduction/36.html">Biological Membranes are determined by The properties of Cell/29.html">The Lipid Bilayer, most of their specific Functions are carried out by Proteins. This is why the types and amounts of proteins in a membrane vary widely: in the myelin membrane, which serves primarily to insulate axons, proteins constitute less than 25% of the membrane mass, whereas in membranes involved in energy Transduction processes (such as the inner membranes of Mitochondria and METABOLISM/14.html">Chloroplasts), they account for about 75% of the membrane mass. In a typical Plasma Membrane, proteins make up approximately half of its mass, which is midway between these two extremes. Because the size of a lipid molecule is very small compared to that of a protein molecule, it can be concluded that there are always far more lipid molecules than protein molecules in a membrane. For example, if proteins make up 50% of the membrane mass, there are approximately 50 lipid molecules for every protein molecule.

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6.2.1. The polypeptide chain of many Membrane Proteins spans the lipid bilayer once or multiple times [8]

Many membrane proteins extend entirely across the bilayer (Examples 1 and 2 in Fig. 6-14). Like their lipid neighbors, these so-called transmembrane proteins are amphipathic: they have hydrophobic regions that pass through the membrane and interact with the hydrophobic tails of lipid molecules inside the bilayer, and hydrophilic regions exposed to Water on both sides of the membrane. The Structure/106.html">Hydrophobicity of some membrane proteins is increased by the covalent attachment of a fatty acid chain that inserts into the bilayer from its cytoplasmic side (examples 1 and 2 in Fig. 6-14). Some intracellular membrane proteins are attached to the bilayer solely by a fatty acid chain (example 3 in Fig. 6-14), and There are also surface proteins associated with the bilayer via covalent interactions (through a specific oligosaccharide) with phosphatidylinositol, a minor phospholipid located in the outer lipid monolayer of the plasma membrane (example 4 in Fig. 6-14).

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Fig. 6-14. Five ways in which membrane proteins associate with the lipid bilayer. Transmembrane proteins span the bilayer as a single α-Helix (1) or multiple α-helices (2). Some of these proteins (1 and 2) have a covalently attached fatty acid chain embedded in the cytosolic monolayer (1). Other membrane proteins associate with the bilayer solely via a covalently attached lipid—either a fatty acid chain embedded in the cytosolic monolayer (3) or, much less commonly, via the phospholipid phosphatidylinositol, embedded in the outer monolayer and linked to the protein through an oligosaccharide (4). Finally, many proteins associate with the membrane only through noncovalent interactions with other membrane proteins (5). Details are discussed in Chapter 8.

Fig. 6-15. A segment of a transmembrane polypeptide chain spanning the lipid bilayer as an α-helix (based on X-ray crystallographic analysis of a membrane protein crystal). Only the backbone model of the polypeptide chain is shown. Hydrophobic Amino Acids are highlighted in color. The protruding nonpolar side chains of amino acid residues (not shown) interact with the hydrophobic fatty acid chains inside the lipid bilayer. Polar peptide groups form Hydrogen Bonds with one another (not shown) and are thus shielded from the hydrophobic environment of the bilayer. A fragment of a polypeptide from the bacterial photosynthetic reaction center shown in Fig. 6-32 is presented. (J. Deisenhofer et al., Nature 318, 618-624, 1985 and H. Michel et al., EMBO J., 5, 1149-1158, 1986.)

Fig. 6-16. A typical single-pass transmembrane glycoprotein. Note that the polypeptide chain spans the lipid bilayer as a right-handed α-helix, and that oligosaccharide groups and Disulfide Bonds do not form in the cytosolic domain due to the reducing environment of The Cell Cytosol.

Some membrane-associated proteins do not interact with the hydrophobic interior of the lipid bilayer at all. Instead, they are bound to one side of the membrane or the other through noncovalent interactions with other membrane proteins (example 5 in Fig. 6-14). Many of these can be released from the membrane under relatively mild conditions, such as extraction with solutions of very high or low Ionic strength or extreme pH, which disrupt Protein-Protein Interactions but leave the lipid bilayer intact. Such proteins are called peripheral membrane proteins. In contrast, transmembrane proteins, phosphatidylinositol-linked proteins, and some proteins held in the bilayer by a fatty acid chain (as well as other tightly bound proteins that can be released only after disrupting the bilayer with detergents or organic Solvents) are called integral membrane proteins.

The portion of the polypeptide chain of transmembrane proteins that is embedded in the hydrophobic environment of the lipid bilayer consists mainly of amino acid residues with nonpolar side chains. However, because peptide bonds are polar and water molecules are excluded, all peptide bonds in the bilayer tend to form hydrogen bonds with one another (see Fig. 3-26). The number of hydrogen bonds between peptide bonds is maximized if the segment of the polypeptide chain passing through the bilayer forms a regular α-helix. This is how most polypeptide chains cross the membrane (Fig. 6-15). In cases where multiple segments of the polypeptide chain pass through the bilayer, the peptide bonds can, in principle, be fully hydrogen-bonded if these segments are organized as β-sheets. However, more commonly, the polypeptide chain of multipass membrane proteins forms a series of α-helices rather than β-sheets (example 2 in Fig. 6-14). The strict requirement to maximize the number of hydrogen bonds in the absence of water molecules (the prohibition of dehydration) also means that a membrane-spanning polypeptide chain is unlikely to change its direction before fully crossing the membrane, as a bend in the chain would decrease the number of regular hydrogen bonds. Apparently for this reason, no membrane proteins have yet been found that are only partially embedded in a membrane monolayer.

Transmembrane proteins always have a unique orientation in the lipid bilayer. This reflects the asymmetric nature of their Biosynthesis and insertion into the lipid bilayer of The Endoplasmic reticulum, as well as the distinct Functions of the cytosolic and extracellular domains of these proteins. The vast majority of transmembrane proteins are glycosylated. As with Glycolipids, oligosaccharide chains are always present on the extracellular side of the membrane, because sugar residues are added in the lumen of the Endoplasmic reticulum and the Golgi apparatus. Another Asymmetry lies in the protein sulfhydryl (SH) groups, which remain reduced (as cysteines) in cytosolic domains, but are often used to form intra- or interchain disulfide (S—S) bonds in extracellular domains (Fig. 6-16).

6.2.2. Membrane Proteins can be solubilized and purified in detergent solutions [9]

As a rule, transmembrane proteins (and some others tightly associated with the membrane) can be solubilized only using agents that disrupt hydrophobic interactions and, ultimately, the bilayer itself. This is most successfully achieved using detergents—small amphipathic molecules that tend to form micelles in water (Fig. 6-17). When a detergent is mixed with a membrane, the hydrophobic ends of its molecules bind to the hydrophobic regions of membrane proteins, displacing lipid molecules. Because the opposite end of the detergent molecule is polar, this binding brings the membrane proteins into solution as detergent-Protein Complexes. Some lipid molecules that are tightly bound to the proteins also remain in these complexes (Fig. 6-18). The polar ends of detergents can be either charged (ionic), as in sodium dodecyl sulfate (SDS), or uncharged (nonionic), as in Tritons. The structures of these two widely used detergents are shown in Fig. 6-19.

A strong ionic detergent like SDS can solubilize even the most hydrophobic membrane proteins. This allows such proteins to be analyzed by SDS-Polyacrylamide gel Electrophoresis. The Development of this technique revolutionized The Study of membrane proteins. Detergents of this type unfold (denature) the polypeptide chain of the protein by inserting into its internal "Hydrophobic core" (see Fig. 3-22). Usually, this causes the proteins to lose their activity, making them unsuitable for functional studies. Nevertheless, proteins can be easily purified in their SDS-denatured form. In some cases, removing the detergent leads to renaturation and recovery of functional activity.

Less hydrophobic membrane proteins can be solubilized at low concentrations of a mild detergent. This allows them to be obtained in a functionally active, if not completely native, form. If the detergent is subsequently removed in the absence of Phospholipids, the membrane proteins usually aggregate and precipitate out of solution (Fig. 6-20). However, if the purified proteins are mixed with phospholipids before the detergent is removed, the proteins in their active form will typically integrate into the lipid bilayer formed by the phospholipid molecules (Fig. 6-21). In this way, functionally active membrane protein systems can be reconstituted from purified components. This is one of the key approaches to studying their functional activity. For example, if a purified protein can be shown to pump ions across a synthetic lipid bilayer in the absence of other proteins, it can be definitively identified as an ion pump. Furthermore, by controlling the reaction conditions in experiments (such as the availability of ATP and ions to the proteins), the detailed MECHANISM OF ACTION of this protein can be elucidated (see Section 6.4.4).

Fig. 6-17. Cytology/practical/72.html">Cross section of a micelle formed in water by detergent molecules. Detergent molecules are amphipathic, having both a polar and a nonpolar end.

Fig. 6-18. Solubilization of membrane proteins with detergent. The detergent disrupts the lipid bilayer, bringing the proteins into solution as complexes with lipid and detergent molecules. Membrane phospholipids are also solubilized by the detergent.

Fig. 6-19. Structures of the molecules of two widely used detergents: sodium dodecyl sulfate (SDS, an anionic detergent) and Triton X-100 (a nonionic detergent). Note that the bracketed region is repeated seven times.

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6.2.3. The surface of membrane proteins facing the Cytoplasm can be studied using erythrocyte "ghosts" [10]

Much more is known about the plasma membrane of human erythrocytes (Fig. 6-22) than about any other Eukaryotic Cell membrane. This situation has arisen for several reasons. 1) Erythrocytes can be obtained in large quantities (for example, from Blood banks). At the same time, they are practically uncontaminated by other cell types. 2) Since erythrocytes lack a Nucleus and internal Organelles, their plasma membrane is the only membrane of these Cells and can be isolated in pure form, free from contamination by internal membranes. Meanwhile, obtaining the plasma membrane from other cell types, where it usually accounts for less than 5% of the total membrane mass (see Table 8-2), poses a serious problem. 3) Erythrocyte membranes, or "ghosts" (empty shells), are easily obtained by placing the cells in a hypotonic salt solution. The salt concentration in such a solution is lower than in the cell, so water rushes into the erythrocytes, causing them to swell and burst (lysis), releasing Hemoglobin (the major non-membrane protein). 4) Membrane "ghosts" can be studied either in a disrupted state (in which case Reagents interact with molecules on both sides of the membrane) or after spontaneous resealing, when water-soluble reagents cannot penetrate the internal space. In addition, sealed, inside-out vesicles can be prepared from erythrocyte ghosts (Fig. 6-23); this makes it possible to study the extracellular and intracellular (cytoplasmic) sides of the membrane independently of each other. The Use of leaky and sealed erythrocyte ghosts first established that some membrane proteins span the lipid bilayer (see below) and that the Lipid Composition is different on the Two Sides of the bilayer. As with most of the General Principles originally established in studies of erythrocyte membranes, these findings have been gradually confirmed for the membranes of nucleated cells.

The orientation of a particular protein in the membrane can be determined in several ways. One method is based on the fact that water-soluble reagents labeled with fluorescent Dyes or radioactive isotopes cannot penetrate the membrane and therefore covalently bind to specific groups only on its outer surface. The membranes are then solubilized, the proteins are separated by polyacrylamide gel electrophoresis, and the labeled proteins are identified either by radioactivity (gel autoradiography) or by fluorescence under ultraviolet light. Using such vector labeling, one can determine how a specific protein (a band in the gel) is oriented in the membrane: if it is labeled both on the extracellular side (using intact cells and sealed ghosts) and on the cytoplasmic side (using sealed inside-out vesicles), it is undoubtedly a transmembrane protein. An alternative approach is to treat either the outer or inner surface of the membrane with non-penetrating Proteolytic Enzymes: if a protein is partially digested in both cases, it must be a transmembrane protein. In addition, labeled Antibodies can be used to determine which side of the membrane a specific part of a transmembrane protein is located on.

Fig. 6-20. Diagram showing that when detergent is removed from membrane proteins solubilized with it, the hydrophobic regions on the protein surface, which are virtually unprotected from contact with water, tend to interact with one another, resulting in The formation of large protein aggregates that precipitate out of solution.

Fig. 6-21. Solubilization, purification, and Reconstitution of functional (Na⁺ + K⁺)-ATPase molecules in phospholipid vesicles. (Na⁺ + K⁺)-ATPase is a cation pump present in the Plasma Membranes of most animal cells. It uses the energy of ATP Hydrolysis to pump Na⁺ ions out of the cell and K⁺ into the cell. Reconstitution is carried out in the presence of high concentrations of Na⁺ and ATP, as the ATPase functions as a pump only when There is a sufficient concentration of these substances inside the vesicles. The detergent is removed by prolonged dialysis or Chromatography.

Fig. 6-22. Scanning electron micrograph of human erythrocytes. The cells are biconcave in shape and lack nuclei. (Courtesy of Bernadette Chailley.)

Fig. 6-23. Preparation of leaky and sealed erythrocyte ghosts, as well as sealed right-side-out and inside-out vesicles. Erythrocytes are shown to burst at only one site, yielding ghosts with a single hole. Small vesicles are produced by mechanical disruption of the ghosts. The orientation of the membranes in the vesicles is determined by the ionic conditions during the disruption Procedure.

Fig. 6-24. SDS-polyacrylamide-gel electrophoresis pattern of the proteins in the human erythrocyte membrane. The gel is stained with Coomassie blue (A). The positions of some of the proteins in the gel (B); the band corresponding to Glycophorin is highlighted in color to facilitate its identification near band 3. Other bands in the gel are omitted from the drawing. The abundant carbohydrate residues on glycophorin retard the migration of this protein so that it runs almost as slowly as the much larger band 3 molecules. (Courtesy of Ted Steck.)

When the plasma membrane proteins of human erythrocytes are studied by SDS-PAGE, about 15 major proteins can be identified, with molecular weights ranging from 15,000 to 250,000. Three of these—spectrin, glycophorin, and the so-called band 3—together account for more than 60% (by weight) of the total membrane protein (Fig. 6-24). All three proteins are associated with the membrane in different ways. Therefore, we will consider these proteins as examples of the three major Modes of Protein-membrane association.

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6.2.4. Spectrin is a cytoskeletal protein noncovalently associated with the cytoplasmic side of The erythrocyte membrane [11]

Most of the membrane proteins of human erythrocytes are peripheral membrane proteins associated with the bilayer on its cytoplasmic side. The most abundant of these proteins, spectrin, is a long, thin, flexible rod about 100 nm in length. It accounts for about 25% of the membrane protein mass, corresponding to 2.5 x 10⁵ copies per cell. Spectrin is a major component of the protein meshwork (Cytoskeleton) that maintains the structural integrity and biconcave shape of erythrocytes (see Fig. 6-22). If the cytoskeleton is extracted from erythrocyte ghosts with low-ionic-strength solutions, the membrane fragments into small vesicles.

Fig. 6-25. Spectrin molecules from human erythrocytes. A. Schematic diagram. B. Electron micrograph. Each heterodimer consists of two antiparallel, slightly helical, flexible polypeptide chains that interact noncovalently at multiple points, including both ends. The phosphorylated "HEAD" region, where two dimers associate to form a tetramer, is on the left.

Each of the alpha and beta chains consists of A large number of repeating domains, 106 amino acid residues in length. Each domain is thought to be organized into a group of three alpha-helices (not shown) connected by nonhelical loops. (B) shows platinum-shadowed spectrin molecules. (A—from D. W. Speicher and V. T. Marchesi, Nature 311, 177-180; B—courtesy of D. M. Shotton et al., J. Mol. Biol., 131, 303-329, 1979. Academic Press Inc. (London) Ltd.)

The spectrin molecule consists of two large polypeptide chains: alpha-spectrin (about 240,000 daltons) and beta-spectrin (about 220,000 daltons). Apparently, each chain is made up of many alpha-helical segments, grouped in threes and linked by non-helical regions (Fig. 6-25). These spectrin heterodimers spontaneously self-associate (head-to-head) to form 200-nm-long tetramers. The ends of five or six tetramers are linked together by binding to short Actin filaments and another protein (band 4.1) in what is called a "junctional complex." This forms a flexible, netlike meshwork on the cytoplasmic surface of the membrane (Fig. 6-26). It is this spectrin-based cytoskeleton that enables erythrocytes to withstand the shear stress on their membrane as they pass through narrow capillaries. In anemic mice and humans with hereditary spectrin abnormalities, the erythrocytes are spherical (rather than biconcave) and abnormally fragile. The severity of the anemia is directly proportional to the degree of spectrin deficiency.

By binding radiolabeled spectrin to erythrocyte membranes from which spectrin and some other peripheral proteins had been previously removed, it was possible to identify the protein responsible for anchoring the spectrin cytoskeleton to the plasma membrane. This large intracellular protein was named ankyrin. It binds both to beta-spectrin and to the cytoplasmic domain of the transmembrane protein band 3 (see Fig. 6-26). By linking band 3 to spectrin, ankyrin anchors the spectrin meshwork to the membrane. This greatly reduces The rate of diffusion of band 3 protein molecules in the lipid bilayer. However, the spectrin-based cytoskeleton can also connect to the membrane via another mechanism. The cytoskeletal protein band 4.1 (which binds spectrin and actin) has been shown to connect to the cytoplasmic domain of glycophorin, another transmembrane erythrocyte protein.

Fig. 6-26. Schematic diagram (A) and electron micrograph (B) of the spectrin-based cytoskeleton on the cytoplasmic surface of the human erythrocyte membrane. The structure shown in (A) is derived mainly from in vitro studies of interactions between purified proteins. Spectrin dimers associate head-to-head to form tetramers, which are linked together by short actin filaments (containing 15 monomers) and band 4.1 protein (two or three other proteins are not shown) to form a meshwork. This cytoskeletal network is anchored to the membrane through the interaction of spectrin tetramers with band 3 protein molecules, not directly, but via ankyrin molecules. It can also bind to the membrane through the interaction of band 4.1 protein with glycophorin molecules (not shown). The electron micrograph shows erythrocytes after fixation and negative staining. The spectrin network has been intentionally stretched to reveal individual structural details; in a normal cell, this network occupies only 1/10 of the area shown. (Courtesy of T. Byers and D. Branton, Proc. Natl. Acad. Sci. USA, 82, 6153-6157, 1985.)

Similar, but much more sophisticated and complex cytoskeletal networks underlie the plasma membranes of nucleated cells. These networks, which make up the cortical regions (or cortex) of the cytoplasm, contain numerous actin filaments that appear to be linked to plasma membranes in several different ways. Proteins structurally homologous to spectrin, ankyrin, and band 4.1 have been found in the cortex, but their Organization and Functions are still not fully understood.

6.2.5. Glycophorin spans the lipid bilayer as a single alpha-helix [11]

Glycophorin is one of the two major proteins exposed on the outer surface of human red Blood Cells. It was the first membrane protein for which the complete Amino Acid Sequence was determined. Glycophorin is a small transmembrane glycoprotein (131 amino acid residues). Most of the mass of this protein is located on the outer surface of the membrane, where its hydrophilic N-terminal domain is also localized. Fifteen separate oligosaccharide side chains are attached to this region of the protein molecule, containing a total of about 100 sugar residues, which accounts for approximately 60% of the mass of the glycoprotein molecule. In fact, glycophorin molecules carry the vast majority of cell-surface CARBOHYDRATES (including more than 90% of the sialic acid) and, consequently, most of the negative charge on the cell surface. The hydrophilic C-terminal tails of these molecules are submerged in the cytosol, while a hydrophobic alpha-helical segment of approximately 20 amino acid residues spans the lipid bilayer.

Although cells contain many glycophorin molecules (more than 6 x 10⁵), their function remains unknown. Moreover, individuals whose red blood cells lack most of these molecules appear to be perfectly healthy. Glycophorin is found only in red blood cells; however, structurally, it can be classified under the general class of membrane Glycoproteins that span the lipid bilayer as a single alpha-helix (see example 1 in Fig. 6-14 and Fig. 6-16). Various cell-surface receptors belong to this very class of proteins.

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6.2.6. Band 3 of the human erythrocyte membrane is an anion-transport protein [13]

Unlike glycophorin, the band 3 protein is known to play an important role in cell function. This protein is called band 3 because, during SDS-polyacrylamide gel electrophoresis, it occupies a corresponding position relative to other proteins (see Fig. 6-24). Like glycophorin, band 3 is a transmembrane protein. However, unlike glycophorin, this protein has a globular conformation, and its polypeptide chain (about 930 amino acid residues long) crosses the bilayer at least 10 times. Each red blood cell contains about 10⁶ molecules of band 3 protein, which appear to form dimers and possibly tetramers in the membrane.

The primary function of red blood cells, as is well known, is to transport O₂ from the Lungs to all Tissues and CO₂ from the tissues to the lungs. The band 3 protein is involved in this exchange. In the lungs, red blood cells release the CO₂ accumulated in the tissues by exchanging HCO₃^- ions for Cl^-. The membrane contains a specialized anion transport protein to facilitate this process. Gas exchange can be blocked by a specific inhibitor that binds to the transport protein. Using radioactively labeled inhibitors, this anion transport protein was identified as the band 3 protein. More recently, the anion transport process was reconstituted in vitro using purified band 3 protein incorporated into phospholipid vesicles. Very similar anion transport proteins have been found in many other nucleated cells, where they help control intracellular pH.

The band 3 protein can be visualized as intramembrane particles using freeze-fracture Electron Microscopy. In this technique, cells are frozen in liquid nitrogen, and the resulting block of ice is fractured with a sharp blade. The fracture plane typically passes through the hydrophobic core of the membrane bilayer, splitting it into two monolayers. Platinum is then shadowed onto the exposed surfaces, and the resulting platinum replicas are examined under an Electron microscope. This technique reveals (Fig. 6-27) two distinct hydrophobic inner surfaces: that of the cytoplasmic (or protoplasmic) half of the bilayer (P-face) and that of the extracellular half of the bilayer (E-face, from external). In such preparations, human erythrocyte membranes appear studded with intramembrane particles of relatively homogeneous size (7.5 nm in diameter). They are found to be more numerous on the P-face than on the E-face (Fig. 6-28). These are likely mostly band 3 proteins, as similar particles are observed upon fracturing synthetic lipid bilayers reconstituted with band 3 protein. Fig. 6-29 demonstrates that it is indeed the band 3 protein molecules, rather than glycophorin molecules, that are visible in freeze-fracture ELECTRON MICROGRAPHS OF red blood cell membranes. After all, it is easy to imagine how a transmembrane protein, such as band 3, the bulk of which is located within the lipid bilayer, can mediate the passive transport of polar molecules across a nonpolar bilayer. Clearly, the band 3 protein (or its dimer or tetramer) can provide a transmembrane hydrophilic pathway through which Cl^- and HCO^-₃ ions are transported without contacting the hydrophobic environment of the lipid bilayer (see Fig. 6-43, A). It is highly unlikely that such transport could be carried out by a glycophorin molecule, which spans the bilayer as a simple alpha-helix. To understand The Mechanism of membrane transport proteins, precise information about their three-dimensional structure within the bilayer is required. The first transport protein for which such details became known was Bacteriorhodopsin—a protein that functions as a light-driven proton (H⁺) pump in the plasma membrane of certain Bacteria. The structure of bacteriorhodopsin is similar to that of other membrane proteins. This protein deserves a more detailed Discussion.

Fig. 6-27. Diagram showing how freeze-fracture electron microscopy can be used to image the inner Hydrophobic surface of the cytoplasmic (or protoplasmic) half of the bilayer (called the P-face) and the outer half of the bilayer (called the E-face). After the fracturing process shown here, the exposed fracture faces are shadowed with platinum and carbon, the organic material is dissolved away, and the remaining platinum replica is examined in an electron microscope (see also Fig. 4-23).

Fig. 6-28. Freeze-fracture electron micrograph of human red blood cell membranes. Note that the density of intramembrane particles is higher on the cytoplasmic (P) face than on the extracellular (E) face. (Courtesy of L. Engstrom and D. Branton.)

Fig. 6-29. Schematic diagram showing what may happen to glycophorin and band 3 protein molecules in human erythrocyte membranes during the freeze-fracture procedure. When the lipid bilayer is split, either the inner or the outer half of a transmembrane protein is pulled out of the frozen monolayer. Proteins tend to remain with the half of the bilayer that is associated with the larger part of the protein molecule. Therefore, band 3 protein molecules usually remain with the inner (P) fracture face. Because a relatively large portion of the protein is exposed above the fracture face, they are visible in micrographs as intramembrane particles. Glycophorin molecules usually remain with the outer (E) fracture face, but their exposed cytoplasmic tails are too small to create any clearly distinguishable particles.

6.2.7. Bacteriorhodopsin is a proton pump that spans the bilayer as seven alpha-helices [14]

The "purple membrane" of the bacterium Halobacterium halobium is a well-defined, irregularly shaped patch on the plasma membrane (Fig. 6-30) that contains molecules of a single protein, bacteriorhodopsin. Each molecule contains a single light-absorbing prosthetic group, or chromophore (called retinal), which is related to vitamin A and is identical to the chromophore found in the rhodopsin of vertebrate retinal rods (see Section 19.6.6). Retinal is covalently linked to a Lysine side chain of the protein. Upon activation by a single quantum of light, the excited chromophore induces Conformational Changes in the protein, resulting in The transfer of two protons from the inside of the cell to the outside. This translocation generates a proton gradient and an electrical potential gradient across the membrane, which in turn drive the synthesis of ATP by a second protein in the cell's plasma membrane.

Bacteriorhodopsin molecules form a flat crystalline lattice in The cell membrane, resembling a two-dimensional crystal. A combination of low-intensity electron microscopy and electron diffraction has allowed the Determination of the protein's three-dimensional structure and its orientation in the membrane at a resolution of 0.7 nm. The latter method is analogous to X-ray crystallography, which is used to study three-dimensional crystals of soluble proteins. Studies of bacteriorhodopsin have shown that its molecule consists of seven alpha-helices (each containing about 25 amino acid residues) closely packed together (Fig. 6-31). These helices cross the lipid bilayer at approximately right angles to the membrane plane. It is highly probable that protons pass through the membrane with the assistance of the chromophore along a pathway of interacting side chains of the alpha-helices, although the detailed mechanisms of this process are not yet fully understood.

Bacteriorhodopsin belongs to a family of membrane proteins with similar structures but different functions. For example, the photoreceptor protein rhodopsin in vertebrate retinal rods and several other cell-surface receptor proteins that bind specific Hormones are also folded into seven transmembrane alpha-helices. These proteins function as signal transducers rather than transport proteins, as each of them, in response to an extracellular signal, activates another plasma membrane protein that generates a chemical signal in the cytosol.

To fully understand the mechanisms of bacteriorhodopsin function, it is necessary to determine the exact position of all its atoms using X-ray crystallography of protein crystals. However, due to the amphipathic nature of membrane proteins, they are extremely difficult to crystallize. This was first achieved in 1985 for the photosynthetic reaction center of bacteria using X-ray crystallography. For the first time, it was shown how multiple Polypeptides can associate in the membrane to form a complex protein machine.

Fig. 6-30. Schematic drawing of the bacterium Halobacterium halobium, showing patches of purple membrane containing bacteriorhodopsin molecules. These bacteria, which live in salt lakes and receive abundant sunlight, have evolved a variety of light-activated proteins, including bacteriorhodopsin, a light-driven proton pump of the plasma membrane.

Fig. 6-31. Structure of a bacteriorhodopsin molecule and its orientation in the lipid bilayer. The polypeptide chain crosses the bilayer as seven alpha-helices. (After R. Henderson and R.N.T. Unwin, Nature, 257, 28-32, 1975.)

6.2.8. Four different polypeptide chains in a membrane-bound complex form the photosynthetic reaction center in bacteria [15]

In Chapter 3, we discussed how different polypeptides associate to form large multi-enzyme complexes that catalyze complex reactions with high efficiency through the cooperative action of their subunits. Similar protein complexes are found in membranes. The most thoroughly studied of these is the bacterial photosynthetic reaction center. This protein complex is located in the plasma membrane of the purple photosynthetic bacterium Rhodopseudomonas viridis. It uses absorbed light energy to generate a high-energy electron, allowing it to cross the membrane in less than a nanosecond. The electron is then transferred to other electron carriers in the membrane, which use part of The energy released during electron transport to synthesize ATP in the cytosol. The reaction center is constructed from four different polypeptides: L, M, H, and cytochrome. To study its three-dimensional structure, this complex was solubilized in a detergent solution, crystallized as a protein-detergent complex, and analyzed by X-ray crystallography. The reaction center was found to contain four chlorophyll molecules and eight other electron-transporting Cofactors. In Chapter 7, we will discuss how determining the exact position of each cofactor within the complex proved crucial for understanding Photosynthesis. No less significant (and highly relevant to the topic of this chapter) was the elucidation of the ORGANIZATION OF THE four protein subunits within the transmembrane complex. The L and M subunits are homologous, each consisting of five alpha-helices that span the lipid bilayer of the plasma membrane (Fig. 6-32). These two subunits form a heterodimer that constitutes the core of the reaction center, with its 10 alpha-helices surrounding the electron carriers. The H subunit has only a single transmembrane alpha-helix, while the remainder of its polypeptide chain is folded into a globular domain on the cytoplasmic side of the membrane, where it is bound to the L-M heterodimer. Cytochrome is a peripheral membrane protein associated with the L-M heterodimer on the extracellular side of the membrane (see Fig. 6-32).

Fig. 6-32. STRUCTURE OF THE photosynthetic reaction center of the bacterium Rhodopseudomonas viridis, based on X-ray crystallographic Analysis of the transmembrane protein complex. The protein complex consists of four subunits: L, M, H, and cytochrome. Subunits L and M form the core of the reaction center. Each of the L and M subunits contains 5 α-helices spanning the bilayer. The positions of the electron-transport Coenzymes are shown in color. (Drawn by J. Richardson, based on Deisenhofer et al., Nature, 318, 618-624, 1985.)

The two 'additional' subunits (H and cytochrome) greatly increase the efficiency of the photosynthetic reaction, which is essentially catalyzed by the L-M heterodimer: cytochrome supplies the heterodimer with electrons, while the H subunit presumably links the reaction center to a variety of other proteins. The L-M heterodimer has proven to be highly conserved in evolution; apparently, a pair of closely related proteins forms the core of one of the photosynthetic reaction centers in green plants.

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6.2.9. Many membrane proteins diffuse in the plane of the membrane [16]

Like Membrane Lipids, membrane proteins cannot flip from one side of the bilayer to the other (a process known as flip-flop), but they can rotate around an axis perpendicular to the plane of the bilayer (rotational diffusion). In addition, many membrane proteins can move laterally within the plane of the membrane (lateral diffusion). That some plasma membrane proteins are capable of moving in the plane of the bilayer was first demonstrated directly in 1970 through experiments on hybrid cells (heterokaryons) produced artificially by fusing mouse and human cells. To distinguish between mouse and human plasma membrane proteins, two groups of labeled antibodies were used. Initially, the mouse and human proteins were confined to their respective halves of the heterokaryon. However, within about half an hour, both sets of proteins had diffused and spread over the entire cell surface (Fig. 6-33). Even more convincing Evidence for the mobility of membrane proteins came from the discovery of a process called patching (see Section 6.5.13). The Essence of this phenomenon is as follows: if cell-surface proteins are cross-linked with antibodies, they aggregate into large clusters, forming discrete, irregularly shaped zones (patches) on the cell surface. However, for antibodies to cross-link membrane proteins into large complexes, these proteins must be free to move in the plane of the bilayer.

Fig. 6-33. Diagram of an experiment demonstrating the mixing of plasma membrane proteins in mouse-human cell hybrids. Mouse and human proteins are initially located on their respective halves of the newly formed plasma membrane of the heterokaryon, but mix over time. Two Types of antibodies, labeled with different ligands, were used to visualize the proteins. (Under a fluorescence microscope, fluorescein appears green and rhodamine appears red.) (From L. D. Frye and M. Edidin, J. Cell Sci., 7, 319-335, 1970, with permission of The Company of Biologists.)

The rate of lateral diffusion can be measured using the technique of fluorescence recovery after photobleaching (FRAP). This method was first used to measure the diffusion rate of individual rhodopsin molecules in the disc membranes of vertebrate retinal rod cells. As mentioned earlier, rhodopsin has a structure similar to that of bacteriorhodopsin and contains the same retinal chromophore group. The diffusion of rhodopsin molecules can be measured as follows. In rhodopsin molecules located on one side of the rod, the chromophore is bleached using a highly focused, high-intensity light beam, and then the time required for the bleached molecules to mix with unbleached ones via diffusion is measured (Fig. 6-34). The diffusion rate (or diffusion coefficient, D) was found to be approximately 5 x 10^-9 cm² s^-1. This is about half the diffusion coefficient of phospholipid molecules in the membrane (see Section 6.1.2) and, at the same time, the highest coefficient among all known membrane proteins.

The same technique has been used to study membrane proteins that do not contain chromophores. First, fluorescent ligands were attached to these proteins. Typically, fluorescein-labeled monovalent antibodies (i.e., antibody fragments with a single antigen-binding site, which are therefore unable to cross-link neighboring molecules) were used for this purpose. These bound ligands were then bleached with a laser beam, and the time required for membrane proteins carrying unbleached antibodies to diffuse into the bleached area was measured (Fig. 6-35). The diffusion rates of various plasma membrane glycoproteins measured in this way were typically at least 5 to 50 times lower than those of rhodopsin molecules. These relatively low diffusion rates are not intrinsic Properties of Individual glycoprotein molecules, as the same glycoprotein molecules diffuse much faster in reconstituted synthetic bilayers. The true reason for the low diffusion rates of plasma membrane glycoproteins measured by FRAP remains unclear. One possible explanation is that the bulky polysaccharide chains of the extracellular domains of these molecules interact with the oligosaccharide chains of other membrane glycoproteins, resulting in slow diffusion. In at least some cases, removal of carbohydrate chains greatly increased the Rate of protein diffusion.

Fig. 6-34. Measurement of the lateral diffusion rate of rhodopsin molecules in retinal rod disc membranes. The chromophores of rhodopsin molecules are bleached on one side of the cell; the rate at which bleached and unbleached rhodopsin molecules mix by diffusion is then measured. (From M. Poo and R. A. Cone, Nature, 247, 438-441, 1974.)

Fig. 6-35. Measurement of the lateral diffusion rate of a plasma membrane glycoprotein. A. A specific glycoprotein is labeled with a fluorescent monovalent antibody that binds only to this protein. After bleaching the antibodies with a small-diameter laser beam, the recovery of fluorescence intensity is measured as bleached molecules diffuse out of and unbleached molecules diffuse into the irradiated area. B. Graph showing the rate of fluorescence recovery. The larger the diffusion coefficient of the membrane glycoprotein, the faster the recovery occurs.

6.2.10. Cells can confine proteins and lipids to specific membrane domains [17]

A major step forward in understanding membrane Structure and function was the realization that biological membranes are two-dimensional fluids. However, it is clear that the view of the membrane as a lipid sea in which proteins float freely is highly simplified. Many cells have The ability to confine membrane proteins to specific domains within a continuous lipid bilayer. For example, in epithelial cells lining the gut or Kidney tubules, certain plasma membrane enzymes and transport proteins are confined to the apical surface of the cells, while others are restricted to the basolateral surface (Fig. 6-36). This asymmetric distribution of membrane proteins is essential for epithelial function (to be discussed later, see Section 6.4.11). The Lipid Composition of these two membrane domains is also different, indicating that epithelial cells can restrict the diffusion of both protein and lipid molecules between domains (although experiments with labeled lipid molecules suggest that this is true only for lipid molecules in the outer monolayer of the membrane). This spatial segregation of proteins and lipids appears to be maintained (at least in part) by barriers formed by a specialized type of cell junction—tight junctions (see Section 14.1.1). The question of why the membrane proteins that form cell junctions do not move laterally within the interacting membranes (see Fig. 6-38, 5) is discussed below.

Fig. 6-36. Schematic diagram of an epithelial cell showing how the distribution of different proteins in the plasma membrane can be restricted. Proteins A (in the apical membrane) and B (in the basolateral membrane) can diffuse laterally only within their respective membrane domains; their entry into other regions is likely prevented by specialized cell junctions called tight junctions. Lipid molecules in the outer (non-cytoplasmic) monolayer of the plasma membrane are also unable to diffuse between the two domains, whereas lipids in the inner (cytoplasmic) monolayer are free to do so.

Fig. 6-37. Three domains of the guinea pig sperm plasma membrane revealed by Monoclonal Antibodies. The sperm cell is shown schematically at the top of the figure. In each of the three micrographs (A, B, and C), immunofluorescence staining of the cell surface with different monoclonal antibodies is combined with a phase-contrast image of the same cells. Antibodies in (A) label only the anterior head, in (B) only the posterior head, and in (C) only the tail. (A and B courtesy of D. G. Myles et al., Cell, 23, 434-439, 1981. C courtesy of P. Primakoff and D. G. Myles, Dev. Biol., 98, 417-428, 1983.)

Membrane domains can also be maintained by a cell without cell-to-cell junctions. For example, an animal sperm cell is a single cell consisting of two structurally and functionally distinct parts—the head and the tail—covered by a continuous plasma membrane. When sperm cells were examined by immunofluorescence microscopy using various antibodies against cell-surface Antigens, the plasma membrane was found to consist of at least three distinct domains (Fig. 6-37). In some cases, antigens can diffuse within their own confined domains. However, the mechanism by which the segregation of these domains is maintained remains poorly understood.

In the two examples discussed, the diffusion of proteins and lipids was restricted to specialized domains within a continuous plasma membrane. Cells also possess more powerful ways of immobilizing specific membrane proteins. This is well illustrated by the purple membrane of Halobacterium. In this case, bacteriorhodopsin molecules are assembled into large two-dimensional crystals in which individual protein molecules are fixed relative to one another. Large aggregates of this type diffuse very slowly. More generally, the restriction of the lateral mobility of specific membrane proteins is due to their interaction with macromolecular assemblies located outside or inside the cell. We have already mentioned that some red blood cell membrane proteins are tightly linked to the internal cytoskeleton. In other cell types, plasma membrane Proteins can also be linked to the cytoskeleton, the Extracellular matrix, or both. Four known ways of immobilizing specific membrane proteins are shown in Fig. 6-38.

Summary

The lipid bilayer determines the basic structural features of biological membranes, whereas proteins are responsible for most Membrane Functions. They act as specific receptors and enzymes, mediate The transport of various substances across the membrane, and so on. Most membrane proteins span the bilayer as a single α-helix, but some cross the bilayer multiple times as a series of α-helices. Another group of proteins associates with the membrane without spanning the bilayer, instead attaching to one side of the membrane or the other. Many of these proteins are bound by noncovalent interactions to transmembrane proteins, while others are covalently linked to lipid molecules. Most membrane proteins, like lipids, are free to move in the plane of the membrane. On the other hand, cells can both immobilize specific membrane proteins and confine them, as well as lipids, to specialized domains within a continuous lipid bilayer.

Fig. 6-38. Four ways in which the lateral mobility of plasma membrane proteins can be restricted. Proteins can self-assemble into large aggregates (such as bacteriorhodopsin molecules in the purple membrane of Halobacterium) (A); they can be tethered to macromolecular assemblies outside (B) or inside the cell (C); or they can interact with proteins On the surface of another cell (D).