Lodish Molecular Cell Biology 7th_10 Biomembrane Structure

Molecular model of a lipid bilayer with embedded membrane proteins. Integral membrane proteins have distinct exoplasmic, cytosolic, and membrane-spanning domains. Shown here are portions of the insulin receptor, which regulates cell metabolism. [Ramon Andrade 3Dciencia/ Science Photo Library.]

M embranes participate in many aspects of cell structure and function. The plasma membrane defines the cell and separates the inside from the outside. In eukary-otes, membranes also define the intracellular organelles such as the nucleus, mitochondrion, and lysosome. These biomem-branes all have the same basic architecture-a phospholipid bilayer in which proteins are embedded (Figure 10-1). By pre-venting the unassisted movement of most water-soluble sub-stances from one side of the membrane to the other, the phospholipid bilayer serves as a permeability barrier, helping to maintain the 'characteristic differences between the inside and outside of the cell or organelle; in turn, the embedded pro-teins endow the membrane with specific functions, such as regulated transport of substances from one side to the other. Each cellular membrane has irs own set of proteins that allow it to carry out a multitude of different functions.

Prokaryotes, the simplest and smallest cells, are about 1-2 j.J..m in length and are surrounded by a single plasma membrane; in most cases they contain no internal membrane-limited subcompartments (see Figure 1-11 ). However, this

OUTLINE

10.1 The lipid Bilayer: Composit ion and Structural Organization

10.2 Membrane Proteins: Struct ure and Basic Functions

445

455

CHAPTER

Biomembrane Structure

single plasma membrane contains hundreds of different types of proteins that are integral to the function of the cell. Some of these proteins catalyze A TP synthesis and initiation of DNA replication, for instance. Others include the many types of membrane transport proteins that enable specific ions, sugars, amino acids, and vitamins to cross the otherwise impermeable phospholipid bilayer to enter the cell and that allow specific metabolic products to exit. Receptors in the plasma membrane are proteins that allow the cell to recognize chemical signals present in its environment and adjust its metabolism or pattern of gene expression in response.

Eukaryotes also have a plasma membrane studded with a multitude of proteins that perform a variety of functions, in-cluding membrane transport, cell signaling, and connecting cells into tissues. In addition, eukaryotic cells-which are generally much larger than prokaryotes-also have a variety of internal membrane-bound organelles (see Figure 9-32). Each organelle membrane has a unique complement of pro-teins that enable it to carry out its characteristic cellular func-tions, such as ATP generation (in mitochondria) and DNA

10.3 Phospholipids, Sphingolipids, and Cholesterol: Synthesis and Int racellular Movement 464

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FIGURE 10-1 Fluid mosaic model of biomembranes. A bilayer of phospholipids -3 nm thick provides the basic architecture of all cellular membranes; membrane proteins give each cellular membrane its unique set of functions. Individual phospholipids can move laterally and spin within the plane of the membrane, giving the membrane a fluidlike consistency similar to that of olive oil. Noncovalent interactions between phospholipids, and between phospholipids and proteins, lend strength and resilience to the membrane, while the hydrophobic core of the bilayer prevents the unassisted movement of water-soluble

synthesis (in the nucleus). Many plasma membrane proteins also bind components of the cytoskeleton, a dense network of protein filaments that crisscrosses the cytosol to provide mechanical support for cellular membranes, interactions that are essential for the cell to assume its specific shape and for many types of cell movements.

Despite playing a structural role in cells, membranes are nor rigid structures. They can bend and flex in three dimen-sions while still maintaining their integrity, due in part to abundant noncovalent interactions that hold lipids and pro-teins together. Moreover, within the plane of the membrane, there is considerable mobility of individual lipids and proteins. According to the fluid mosaic model of biomembranes, first

Hydrophilic phospholipid head group

Phospholipid bilayer

Hydrophobic fatty acyl side chains

substances from one side to the other.lntegral (transmembrane) proteins span the bilayer and often form dimers and higher-order oligomers. Lipid-anchored proteins are tethered to one leaflet by a covalently attached hydrocarbon chain. Peripheral proteins associate with the membrane primarily by specific noncovaler:lt interactions wit h integral proteins or membrane lipids. Proteins in t he plasma membrane also make extensive contact with the cytoskeleton. [After D. Engelman, 2005, Nature 438:578-580.]

(a)

proposed by researchers in the 1970s, the lipid bi layer behaves (b) in some respects like a two-dimensional fluid, with individual lipid molecules able to move past one another as well as spin in place. Such fluidity and flexibility not only allows organelles to assume their typical shapes, but also enables the dynamic

FIGURE 10-2 Eukaryotic cell membranes are dynamic structures. (a) An electron micrograph of the plasma membrane of an HIV-infected cell, show1ng HIV particles budding into the culture medium. As the virus core buds from the cell, it becomes enveloped by a membrane derived from the cell's plasma membrane that contains specific viral proteins. (b) Stacked membranes of the Golgi complex with budding vesicles. Note the irregular shape and curvature of these membranes. [Part (a) from W. Sundquist and U. von Schwedler, University of Utah; part (b) from Biology Pies/Photo Researchers, Inc.]

444 CHAPTER 10 Biomembrane Structure

property of membrane budding and fusion, such as occurs when viruses are released from an infected cell (Figure 1 0-2a) and when the internal cellular membranes of the Golgi com-plex bud into vesicles in the cytosol (Figure 10-2b) and then fuse with other membranes to transport their contents from one organelle to another (Chapter 14).

We begin our examination of biomembranes by consid-ering their lipid components. These not only affect mem-brane shape and function but also help anchor proteins to the membrane, modify membrane protein activities, and transduce signals to the cytoplasm. We then consider the structure of membrane proteins . Many of these proteins have large segments that are embedded in the hydrocarbon core of the phospholipid bilayer, and we will focus on the principal classes of such membrane proteins. Finally, we consider how lipids such as phospholipids and cholesterol are synthesized in cells and distributed to the many mem-branes and organelles. Cholesterol is an essential component of the plasma membrane of all animal cells but is toxic to the organism if present in excess.

1 0.1 The Lipid Bilayer: Composition and Structural Organization In Chapter 2 we learned that phospholipids are the principal building blocks of biomembranes. The most common phos-pholipids in membranes are the phosphoglycerides (see Fig-ure 2-20), but as we will see in this chapter, there are multiple types of phospholipid. All phospholipids are amphipathic molecules-they consist of two segments with very different chemical properties: a fatty acid-based (fatty acyl) hydrocar-bon "tail" that is hydrophobic and partitions away from water, and a polar "head group" that is strongly hydrophilic, or water loving, and tends to interact with water molecules. The interactions of phospholipids with each other and with water largely determine the structure of biomembranes.

Besides phospholipids, biomembranes contain smaller amounts of other amphipathic lipids, such as glycolipids and cholesterol, which contribute to membrane function in im-portant ways. We first consider the structure and properties of pure phospholipid bilar,ers and then discuss the composi-tion and behavior of natural cell membranes. We will see how the precise lipid composition of a given membrane in-fluences its physical properties.

Phospholipids Spontaneously Form Bilayers The amphipathic nature of phospholipids, which governs their interactions, ts cnrical to the structure of biomembranes. When a suspension of phospholipids is mechanically dispersed in aqueous solution, the phospholipids aggregate into one of three forms: spherical micelles and liposomes, or sheetlike phospholipid bilayers, which arc two molecules thick (Figure 10-3). The type of structure formed by pure phospholipids or

(a) Membrane bilayer

(b) Polar head

(c)

Micelle

Liposome

FIGURE 1 0-3 The bilayer structure of biomembranes. (a) Electron micrograph of a thin section through an erythrocyte membrane stained with osmium tetroxide. The characteristic "railroad track" appearance of the membrane indicates the presence of two polar layers, consistent with the bilayer structure of phospholipid mem-branes. (b) Schematic interpretation ofthe phospholipid bilayer in which polar groups face outward to shield the hydrophobic fatty acyl tails from water. The hydrophobic effect and van der Waals interactions between the fatty acyl tails drive the assembly of the bilayer (Chapter 2). (c) Cross-sectional views of two other structures formed by dispersal of phospholipids in water. A spherical micelle has a hydrophobic interior composed entirely of fatty acyl chains; a sphericalliposome consists of a phospholipid bilayer surrounding an aqueous center. [Part (a) courtesy of J. D. Robertson.]

a mixture of phospholipids depends on several factors, includ-ing the length of the fatty acyl chains in the hydrophobic tail, their degree of saturation (i.e., the number of C-C and C=C bonds), and temperature. In all three structures, the hydro-phobic effect causes the fatty acyl chains to aggregate and

10.1 The Lipid Bilayer: CompositiOn and Structural Organization 445

exclude water molecules from the "core.'' Micelles are rarely formed from natural phospholipids, whose fatty acyl chains generally arc too bulky to fit into the interior of a micelle. However, micelles arc formed if one of the two fatty acyl chains that make up the tail of a phospholipid is removed by hydrolysis, forming a lysophospholipid, as occurs upon treat-ment with the enzyme phospholipase. In aqueous solution, common detergents and soaps form micelles that behave like the balls in tiny ball bearings, thus giving soap solutions thc1r slippery feel and lubricating properties.

Phospholipids of the composition present in cells sponta-neously form symmetric phospholipid bilayers. Each phos-pholipid layer in this lamellar structure is called a leaflet. The hydrophobic fatty acyl cha ins in each leaflet minimize their contact with water by aligning themselves tightly together in the center of the bilayer, forming a hydrophobic core that is about 3-4 nm thick (Figure 10-3b). The close packing of these nonpolar tails is stabilized by van der Waals interac-tions between the hydrocarbon chains. Ionic and hydrogen bonds stabilize the interactions of the phospholipid polar head groups with one another and with water. Electron mi-croscopy of thin membrane sections of cells stained with os-mium tetroxide, which binds strongly to the polar head groups of phospholipids, reveals the bilayer structure (Figure 10-3a). A cross section of a single membrane stained with osmium tetroxide looks like a railroad track: two thin dark lines (the stained head group complexes) with a uniform light space of about 2 nm between them (the hydrophobic tails).

A phospholipid bilayer can be of almost unlimited size-from micrometers (f.Lm) to millimeters (mm) in length or width-and can contain tens of millions of phospholipid molecules. The phospholipid bilayer is the basic structural unit of nearly all biological membranes. Its hydrophobic core prevents most water-soluble substances from crossing from one side of the membrane to the other. Although biomem-branes contain other molecules (e.g., cholesterol, glycolipids, proteins), it is the phospholipid bilayer that separates two aqueous solutions and acts as a permeability barrier. The lipid bilayer thus defines cellular compartments and allows a separation of the cell's interior from the outside world.

Phospholipid Bilayers Form a Sealed Compartment Surrounding an Internal Aqueous Space Phospholipid bilayers can be generated in the laboratory by simple means, using either chemically pure phospholipids or lipid mixtures of the composition found in cell membranes (Figure 10-4 ). Such synthetic bilayers possess three impor-tant properties. First, they are virtually impermeable to water soluble (hydrophilic) solutes, which do not readily diffuse across the bilayer. This includes salts, sugars, and most other small hydrophilic molecules-including water itself. The sec-ond property of the bilayer is its stability. Hydrophobic and van der Waals interactions between the fatty acyl chains maintain the integrity of the interior of the bilayer structure.


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EXPERIMEN . AL FIGUR -4 Formation and study of pure phospholipid bilayers. (Top) A preparation of biological membranes is treated with an organic solvent, such as a mixture of chloroform and methanol (3:1 ), which selectively solubilizes the phospholipids and cholesterol. Proteins and carbohydrates remain in an insoluble residue. The solvent is removed by evaporation. (Bottom left) If the lipids are mechanically dispersed in water, they spontaneously form a liposome, shown in cross section, with an internal aqueous compartment. (Bottom right) A planar bilayer, also shown in cross section, can form over a small hole in a partition separating two aqueous phases; such a system can be used to study the physical properties of bilayers, such as their permeability to solutes.

Even though the exterior aqueous environment can vary widely in ionic strength and pH, Lht: bilayt:r has the strength to retain its characteristic architecture. Third, all phospho-lipid bilayers can spontaneously form sealed closed compart-ments where the aqueous space on the inside is separated from that on the outside. An "edge" of a phospholipid bi-layer, as depicted in Figure 10-3b, with the hydrocarbon core of the bilayer exposed to an aqueous solution, is unstable; the

Mitochondrion

Outer] Mitochondrial Inner membranes

FIGURE 10-5 The faces of cellular membranes. The plasma membrane, a single bilayer membrane, encloses the cell. In this highly schematic representation, internal cytosol (tan) and external environment (white) define the cytosolic (red) and exoplasmic (gray) faces of the bilayer. Vesicles and some organelles have a single membrane and their internal aqueous space (white) is topologically equivalent to the outside of the cell. Three organelles-the nucleus, mitochondrion, and chloroplast (which is not shown)-are enclosed by two membranes separated by a small intermembrane space. The exoplasmic faces of the inner and outer membranes around these organelles border the intermembrane space between them. For simplicity, the hydrophobic membrane interior rs not indicated in this diagram.

~:.-Cf--1--1---Matrix lntermembrane space

Lysosome

Exoplasmic face

I

Cytosolic face

Plasma membrane

lntermembrane space

exposed fatty acyl side chains would be in an energetically much more stable state if they were not adjacent to water molecules but surrounded by other fatty acyl chains (hydro-phobic effect; Chapter 2). Thus in aqueous solution, sheets of phospholipid bilayers spontaneously sea l their edges, forming a spherical bilayer that encloses an aqueous central compartment. The liposome depicted in Figure 10-3c is an example of such a structure viewed in cross section.

This physical chemical property of a phospholipid bi-layer has important implications for cellular membranes: no membrane in a cell can have an "edge" with exposed hydro-carbon fatty acyl chains. All membranes form closed com-

Exoplasmic face

Cytosolic face

Membrane protein

partments, similar in basic architecture to liposomes. Because all cellular membranes enclose an entire cell or an internal compartment, they have an internal face (the surface ori-ented toward the interior of the compartment) and an exter-nal face (the surface presented to the environment). More commonly, we designate the two surfaces of a cellular mem-brane as the cytosolic face and the exoplasmic face. This nomenclature is useful in highlighting the topological equiv-alence of the faces in different membranes, as diagrammed in figures 10-5 and I 0-6. For example, the exoplasmic face of the plasma membrane is directed away from the cytosol, to-ward the extracellular space or external environment, and defines the outer limit of the cell. The cytosolic face of the plasma membrane faces the cytosol. Similarly for organelles and vesicles surrounded by a single membrane, the cytosolic

FIGURE 10-6 Faces of cellular membranes are conserved during membrane budding and fusion. Red membrane surfaces are cytosolic faces; gray are exoplasmic faces. During endocytosis a segment of the plasma membrane buds inward toward the cytosol and eventually pinches off a separate vesicle. During this process the cytosolic face of the plasma membrane remains facing the cytosol and the exoplasmic face of the new vesicle membrane faces the vesicle lumen. During exocytosis an intracellular vesicle fuses with the plasma membrane, and the lumen of the vesicle (exoplasmic face) connects with the extracellular medium. Proteins that span the membrane retain their asymmetric orientation during vesicle budding and fusion; in particular the same segment always faces the cytosol.

10.1 The Lipid Bilayer: Composition and Structural Organization 447

face faces the cytosol. The exoplasmic face is always directed away from the cytosol and in this case is on the inside of the organelle in contact with the internal aqueous space, or lumen. The lumen of these vesicle~ is topologically equiva-lent to the extracellular space, a concept most easi ly under-stood for vesicles that arise by invagination (endocytosis) of the plasma membrane. The external face of the plasma mem-brane becomes the internal face of the vesicle membrane, while in the vesicle the cytosolic face of the plasma mem-brane still faces the cytosol (figure 1 0-6).

Three organelles-the nucleus, mitochondrion, and chloroplast-are surrounded not by a single membrane, but by two. The exoplasmic surface of each membrane faces the space between the two membranes. This can perhaps best be understood by reference to the endosymbiont hypothesis, dis-cussed in Chapter 6, which posits that mitochondria and chloroplasts arose early in the evolution of eukaryotic cells by the engulfment of bacteria capable of oxidative phosphoryla-tion or photosynthesis, respectively (see Figure 6-20).

Natural membranes from different cell types exhibit a variety of shapes, which complement a cell's function. The smooth, flexible surface of the erythrocyte plasma membrane allows the cell to squeeze through narrow blood capillaries (Figure 10-7a). Some cells have a long, slender extension of the plasma membrane, called a cilium or flagellum, which beats in a whiplike manner (Hgure 10-7b). This motion causes fluid to flow across the surface of a sheet of cells, or a sperm cell to swim toward an egg. The differing shapes and properties of biomembranes raise a key question in cell biology, namely how the composition of biological membranes is regulated to establish and maintain the identity of the different membrane structures and membrane-delimited compartments. We return to this question in Section 10.3 and in Chapter 14.

Biomembranes Contain Three Principal Classes of Lipids The term phospholipid is a somewhat generic term, encompass-ing multiple distinct molecules from multiple classes. It refers to any amphipathic lipid with a phosphate-based head group and a two-pronged hydrophobic tail. A typical biomembrane actu-ally contains three classes of amphipathic lipids: phosphoglyc-erides, sphingolipids, and sterols, which differ in their chemical structures, abundance, and functions in the membrane (Figure 10-8). While all phosphoglycerides are phospholipids, only cer-tain sphingolipids are, and no sterols are.

Phosphoglycerides, the most abundant class of phospholip-ids in most membranes, are derivatives of glycerol 3-phosphate (sec Figure 10-Sa). A typical phosphoglyceride molecule con-sists of a hydrophobic tail composed of two fatty acid-based (acyl) chains esterified to the two hydroxyl groups in glycerol phosphate and a polar head group attached to the phosphate group. The two fatty acyl chains may differ in the number of carbons that they contain (commonly 16 or 18) and their degree of saturation (0, 1, or 2 double bonds) . A phospho-glyceride is classified according to the nature of its head group. In phosphatidylcholines, the most abundant phospho-


(a)

(b)

10 1-lm

FIGURE 10-7 Variation in biomembranes in different cell types. (a) A smooth, flexible membrane covers the surface of the discoid erythrocyte cell as seen in this scanning electron micrograph. (b) Tufts of cilia (Ci) project from the ependymal cells that line the brain ventricles. [Part (a) Copyright 0 Omi Kron/Photo Researchers, Inc. Part (b) from R. G. Kessel and R. H. Kardon, 1979, Tissues and Organs: A Text-Atlas of Scanning Electron Microscopy, W. H. Freeman and Company.]

lipids in the plasma membrane, the head group consists of choline, a positively charged alcohol, esterified to the nega-tively charged phosphate. In other phosphoglycerides, an OH-containing molecule su\:h as ethanolamine, serine, or the sugar derivative inositol is linked to the phosphate group. The negatively charged phosphate group and the positively charged groups or hydroxyl groups on the head group inter-act strongly with water. At neutral pH, some phosphoglycer-ides (e.g., phosphatidylcholine and phosphatidylethanolamine) carry no net electric charge, whereas others (e.g., phosphati-dylinositol and phosphatidylserine) carry a single net nega-tive charge. Nonetheless, the polar head groups in all these

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(b) Sphingolipids

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FIGURE 10-8 Three classes of membrane lipids. (a) Most phosphoglycerides are derivatives of glycerol 3-phosphate (red), which contains two esterified fatty acyl chains that constitute the hydropho bic "tail" and a polar head group" esterified to the phosphate. The fatty acids can vary in length and be saturated (no double bonds) or unsaturated (one, two, or three double bonds). In phosphatidylcholinP (PC), the head group is choline. Also shown are the molecules attached to the phosphate group in three other common phosphoglycerides: phosphatidylethanolamine (PE), phosphatidylserine (PS), and phospha-tidylinositol (PI). Plasmalogens contain one fatty acyl chain attached to glycerol by an ester linkage and one attached by an ether linkage; these contain similar head groups as other phosphoglycerides. (b) Sphingolipids are derivatives of sphingosine (red), an amino alcohol with a long hydrocarbon chain. Various fatty acyl chains are connected

to sphingosine by an amide bond . The sphingomyelins (SM), which contain a phosphocholine head group, are phospholipids. Other sphingolipids are glycolipids in which a single sugar residue or branched oligosaccharide is attached to the sphingosine backbone. For instance, the simple glycolipid glucosylcerebroside (GicCer) has a glucose head group. (c) The major sterols in animdls (cholesterol), fung1 (ergosterol), and plants (stigmasterol) differ slightly in structure, but all serve as key components of cellular membranes. The basic structure of sterols is a four-ring hydrocarbon (yellow). Like other membrane lipids, sterols are amphipathic. The single hydroxyl group is equivalent to the polar head group in other lipids; the conjugated ring and short hydrocarbon chain form the hydrophobic tail. [See H. Sprong et al., 2001, Nature Rev. Mol. Cell Bioi. 2:504.]


phospholipids can pack together into the characteristic bi-layer structure. When phospholipases act on phosphoglycer-ides, they produce lysophospholipids, which lack one of the two acyl chains. Lysophospholipids are not only important signaling molecules, released from cells and recognized by specific receptors; their presence can also affect the physical properties of the membranes in which they reside.

The plasmalogens are a group of phosphoglycerides that wntain one fatty acyl chain attached to carbon 2 of glycerol by an ester linkage and one long hydrocarbon chain attached to carbon 1 of glycerol by an ether (C-0-C) rather than an ester linkage. Plasmalogens are particularly abundant in human brain and heart tissue. The greater chemical stability of the ether linkage in plasmalogens, compared to the ester linkage, or the subtle differences in their three-dimensional structure compared with that of other phosphoglycerides may have as yet unrecognized physiologic significance.

A second class of membrane lipid is the sphingolipids. All of these compounds are derived from sphingosine, an amino alcohol with a long hydrocarbon chain, and contain a long-chain fatty acid attached in amide linkage to the sphin-gosine amino group (see Figure 1 0-8b) . Like phosphoglycer-ides, some sphingolipids have a phosphate-based polar head group. In sphingomyelin, the most abundant sphingolipid, phosphocholine is attached to the terminal hydroxyl group of sphingosine (see Figure 10-8b, SM). Thus sphingomyelin is a phospholipid, and its overall structure is quite similar to that of phosphatidylcholine. Sphingomyelins are similar in shape to phosphoglycerides and can form mixed bilayers with them. Other sphingolipids are amphipathic glycolipids whose polar head groups are sugars that are not linked via a phosphate group (and so technically are not phospholipids). Glucosylcerebroside, the simplest glycosphingolipid, con-tains a single glucose unit attached to sphingosine. In the complex glycosphingolipids called gangliosides, one or two branched sugar chains (oligosaccharides) containing sialic acid groups arc attached to sphingosine. Glycolipids consti-tute 2-10 percent of the total lipid in plasma membranes; they are most abundant in nervous tissue.

Cholesterol and its analogs constitute the third important class of membrane lipids, the sterols. The basic structure of sterols is a four-ring isoprenoid-based hydrocarbon. The structures of the principal yeast sterol (ergosterol) and plant phytosterols (e.g., stigmasterol) differ slightly from that of cholesterol, the major animal sterol (see Figure 1 0-Sc). The small differences in the biosynthetic pathways and structures of fungal and animal sterols arc the basis of most antifungal drugs currently in use. Cholesterol, like the two other sterols, has a hydroxyl substituent on one ring. Although cholesterol IS almost entirely hydrocarbon in composition, it is amphipa-thic hecause its hydroxyl group can interact with water. Be-cause it lacks a phosphate-based head group, it is not a phospholipid. Cholesterol is especially abundant in the plasma membranes of mammalian cells but is absent from most prokaryotic and all plant cells. As much as 30-50 per-cent of the lipids in plant plasma membranes consists of cer-tam steroids unique to plants. Berween 50 and 90 percent of


the cholesterol in most mammalian cells is present in the plasma membrane and associated vesicles. Cholesterol and other sterols are roo hydrophobic to form a bilayer structure on their own. Instead, at concentrations found in natural membranes, these sterols must intercalate between phospho-lipid molecules to be incorporated into biomembranes. When so intercalated, sterols provide structural support to mem-branes, preventing too close a packing of the phospholipids' acyl chains to maintain a sgmficant measure of membrane fluidity, and at the same time conferring the necessary rigidity required for mechanical support. Some of these effects can be highly local, as in the case of lipid rafts, discussed below.

In addition to its structural role in membranes, choles-terol is the precursor for several important bioactive mole-cules. They include bile acids, which are made in the liver and help emulsify dietary fats for digestion and absorption in the intestines; steroid hormones produced by cndocri ne cells (e.g., adrenal gland, ovary, testes); and vitamin D produced in the skin and kidneys. Another critical function of choles-terol is its covalent addition to Hedgehog protein, a key sig-naling molecule in embryonic development (Chapter 16).

Most Lipids and Many Proteins Are. laterally Mobile in Biomembranes In the two-dimensional plane of a bilayer, thermal motion permits lipid molecules to rotate freely around their long axes and to diffuse laterally within each leaflet. Because such movements are lateral or rotational, the fatty acyl chains

Heat

Gel-like consistency Fluid like consistency

FIGURE 10-9 Gel and fluid forms of the phospholipid bilayer. (Top) Depiction of gel-to-fluid transition. Phospholipids with long saturated fatty acyl chains tend to assemble into a highly ordered, gel-like bilayer in which there is little overlap of the nonpolar tails in the two leaflets. Heat disorders the nonpolar tails and induces a transition from a gel to a fluid within a temperature range of only a few degrees. As the chains become disordered, the bilayer also decreases in thickness. (Bottom) Molecular models of phospholipid mono layers in gel and fluid states, as determined by molecular dynamics calculations. [Bottom based on H. Heller et al., 1993, J. Phys. Chem. 97:8343.]

remain in the hydrophobic interior of the bilayer. In both natural and artificial membranes, a typical lipid molecule ex-changes places with its neighbors in a leaflet about 10- times per second and diffuses several micrometers per second at 37 oc. These diffusion rates indicate that the bilayer is 100 times more viscous than water-about the same as the viscosity of olive oil. Even though lipids diffuse more slowly in the bi-layer than in an aqueous solvent, a membrane lipid could diffuse the length of a typical bacterial cell ( 1 p m) in only 1 second and the length of an animal cell in about 20 seconds. When artificial pure phospholipid membranes are cooled below 37 C, the lipids can undergo a phase transition from a liquidlike (fluid) state to a gel-like (semisolid) state, analo-gous to the liquid-solid transition when liquid water freezes (Figure 10-9). Below the phase-transition temperature, the rate of d iffusion of the lipids drops precipitously. At usual physiologic temperatures, the hydrophobic interior of natu-ral membranes generally has a low viscosity and a fluidlike consistency, in contrast to the gel-like consistency observed at lower temperatures.

(a) Membrane protein

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(b)

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In pure membrane bilayers (i.e., in the absence of pro-tein), phospholipids and sphingolipids rotate and move lat-erally, but they do not spontaneously migrate, or flip-flop, from one leaflet to the other. The energetic barrier is roo high; migration would require moving the polar head group from its aqueous environment through the hydrocarbon core of the bilayer to the aqueous solution on the other side. Spe-cial membrane proteins discussed in Chapter 11 are required to flip membrane lipids and other polar molecules from one leaflet to the other.

The lateral movements of specific plasma-membrane proteins and lipids can be quantified by a technique called fluorescence recovery after photobleaching (FRAP). Phos-pholipids containing a fluorescent substituent are used to monitor lipid movement. For proteins, a fragment of a monoclonal antibody that is specific for the exoplasmic do-main of the desired protein and that has only a single antigen-binding site is tagged with a fluorescent dye. With this method, described in Figure 10-10, the rate at which mem-brane molecules move-the diffusion coefficient-can be

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EXPERIMENTAL FIGURE 10-10 Fluorescence recovery after photobleaching (FRAP) experiments can quantify the lateral movement of proteins and lipids within the plasma membrane. (a) Experimental protocol. Step D Cells are first labeled with a fluores-cent reagent that binds uniformly to a spPcific membrane lipid or protein. Step f) A laser light is then focused on a small area of the surface, irreversibly bleaching the bound reagent and thus reducing the fluorescence in the illuminated area. Step tl ln t ime, the fluores-cence of the bleached patch increases as unbleached fluorescent surface molecules diffuse into it and bleached ones diffuse outward. The extent of recovery of fluorescence in the bleached patch is

Time (s) proportional to the fraction of labeled molecules that are mobile in the membrane. (b) Results of a FRAP experiment with human hepatoma cells treated with a fluorescent antibody specific for the asialoglycopro-tein receptor protein. The finding that SO percent of the fluorescence returned to the bleacht:!d area indicates that :,o percent of the receptor molecules in the illuminated membrane patch were mobile and SO percent were immobile. Because the rate of fluorescence recovery is proportional to the rate at which labeled molecules move into the bleached region, the diffusion coefficient of a protein or lipid in the membrane can be calculated from such data. [See V.I. Henis et al., 1990, J. Cell Bioi. 111:1409.]


determined, as well as the proportion of the molecules that arc laterally mobile.

The results of FRAP studies with fluorescence-labeled phospholipids have shown that in fibroblast plasma mem-branes, all the phospholipids are freely mobile over distances of about 0.5 IJ-m, but most cannot diffuse over much longer distances. These findings suggest that protein-rich regions of the plasma membrane about 1 IJ-m in diameter separate lipid-rich regions containing the bulk of the membrane phos-pholipid. Phospholipids are free to diffuse within such re-gions but not from one lipid-rich region to an adjacent one. Furthermore, the rate of lateral diffusion of lipids in the plasma membrane is nearly an order of magnitude slower than in pure phospholipid bilayers: diffusion constants of 10 H cm2/s and 10 ~ cm2/s are characteristic of the plasma membrane and a lipid bilayer, respectively. This difference suggests that lipids may be tightly but not irreversibly bound to certain integral proteins in some membranes, as indeed has recently been demonstrated (see discussion of annular phospholipids, below).

Lipid Composition Influences the Physical Properties of Membranes A typical cell contains many different types of membranes, each with unique properties derived from its particular mix of lipids and proteins. The data in Table 10-1 illustrate the variation in lipid composition in different biomembranes. Several phenomena contribute to these differences. For in-stance, the relative abundances of phosphoglycerides and sphingolipids differ between membranes in the endoplasmic

reticulum (ER), where phospholipids are synthesized, and the Golgi, where sphingolipids arc synthesized. The proportion of sphingomyelin as a percentage of total membrane lipid phosphorus is about six times as high in Golgi membranes as it is in ER membranes. In other cases, the movement of mem-branes from one cellular compartment to another can selec-tively enrich certain membranes in lipids such as cholesterol. In responding to differing environments throughout an or-ganism, different types of cells generate membranes with differing lipid compositions. In the cells that line the intesti-nal tract, for example, the membranes that face the harsh environment in which dietary nutrients are digested have a sphingolipid-to-phosphoglyceride-to-cholesterol ratio of 1:1:1 rather than the 0.5:1.5:1 ratio found in cells subject to less stress. The relatively high concentration of sphingolipid in this intestinal membrane may increase its stability because of extensive hydrogen bonding by the free -OH group in the sphingosine moiety (see Figure 10-8).

The degree of bilayer fluidity depends on the lipid compo-sition, the structure of the phospholipid hydrophobic tails, and temperature. As already noted, van der Waals interac-tions and the hydrophobic effect cause the nonpolar tails of phospholipids to aggregate. Long, saturated fatty acyl chains have the greatest tendency to aggregate, pa~king tightly to-gether into a gel-like state. Phospholipids with short fatty acyl chains, which have less surface area and therefore fewer van der Waals interactions, form more fluid bilayers . Like-wise, the kinks in cis-unsaturated fatty acyl chains (Chapter 2) result in their forming less stable van der Waals interactions with other lipids, and hence more fluid bilayers, than do straight saturated chains, which can pack more tightly together.

TABLE 10-1 Major Lipid Components of Selected Biomembranes

Composition (mol %) - ---

---

Source/Location PC PE + PS SM Cholesterol - ---

Plasma membrane (human erythrocytes) 21 29 21 26

.Viyelin membrane (human neurons) 16 37 13 34

Plasma membrane (. coli) 0 85 0 0

Endoplasmic reticulum membrane (rat) 54 26 5 7

Golgi membrane (rat) 45 20 13 13

Inner mitochondrial membrane (rat) 45 45 2 7

Outer mitochondrial membrane (rat) 34 46 2 11

Primary leaflet location Exoplasmic Cyrosolic Exoplasmic Both

PC phosphatidykholme; PE = phospharidylerhanolamme; PS = phosphandylserine; SM sphingomyelin. ~OL:R

. '

,.

(b)

PC and cholesterol SM SM and

cholesterol

FIGURE 10-11 Effect of lipid composition on bilayer thickness and curvature. (a) A pure sphingomyelin (SM) bilayer is thicker than one formed from a phosphoglyceride such as phosphatidylcholine (PC). Cholesterol has a lipid-ordering effect on phosphoglyceride bilayers that increases their thickness, but it does not affect the thickness of the more-ordered SM bilayer. (b) Phospholipids such as PC have a cylindrical shape and form essentially flat mono layers, whereas those with smaller head groups such as phosphatidylethanolamine (PE) have a conical shape. (c) A bilayer enriched with PC in the exoplasmic leaflet and with PE in the cytosolic face, as in many plasma membranes, would have a natural curvature. [Adapted from H. Sprong et al., 2001, Nature Rev. Mol. Cell Bioi. 2:504.]

Cholesterol is important in maintaining the appropriate fluidity of natural membranes, a property that appears to be essential for normal cell gr0wth and reproduction. Cholesterol restricts the random movement of phospholipid head groups at the outer surfaces of the leaflets, but its effect on the move-ment of long phospholipid tails depends on concentration. At cholesterol concentrations present in the plasma membrane, the interaction of the steroid ring with the long hydrophobic tails of phospholipids tends to immobilize these lipids and thus decrease biomembrane fluidity. It is this property that can help organize the plasma membrane into discrete subdomains of unique hptd and protein composition. At lower cholesterol concentrations, however, the steroid ring separates and dis-perses phospho! ipid tails, causing the inner regions of the membrane to become slightly more fluid.

The lipid composition of a bilayer a lso influences its thickness, which in turn may influence the distribution of

other membrane components, such as proteins, in a particu lar membrane. It has been argued that relatively short trans-membrane segments of certain Golgi-resident enzymes (glycosyltransferases) are an adaptation to the lipid composi-tion of the Golgi membrane and contribute to the retention of these enzymes in the Golgi apparatus. The results of bio-physical studie~ on artificial membranes demonstrate that sphingomyelin associates into a more gel-like and thicker bi-layer than phosphoglycerides uo (Figure 10-1 la ). Cholesterol and other molecules that decrease membrane fluidity also in-crease membrane thickness. Because sphingomyelin tails are already optimally stabilized, the addition of cholesterol has no effect on the thickness of a sphingomyelin bilayer.

Another property dependent on the lipid composition of a bilayer is its curvature, which depends on the relative sizes of the polar head groups and nonpolar tails of its constituent phospholipids. Lipids with long tails and large head groups arc cylindrical in shape; those with small head groups are cone shaped (Figure 10-11 b). As a result, bilayers composed of cylindrical lipids are relatively flat, whereas those contain-ing large numbers of cone-shaped lipids form curved bilayers (figure 10-llc). This effect of lipid composition on bilayer curvature may play a role in the formation of highly curved membranes, such as sites of viral budding (see Figure 10-2) and formation of internal vesicles from the plasma mem-brane (see Figure 1 0-6), and in specialized ~table membrane structures such as microvilli. Several proteins bind to the sur-face of phospholipid bilayers and cause the membrane to curve; such proteins are important in formation of transport vesicles that bud from a donor membrane (Chapter 14).

Lipid Composition Is Different in the Exoplasmic and Cytosolic leaflets A characteristic of all biomembranes is an asymmetry in lipid composition across the bilayer. Although most phos-pholipids are present in both membrane leaflets, some are commonly more abundant in one or the other leaflet. For instance, in plasma membranes from human erythrocytes and Maclin Darby canine kidney (MDCK) cells grown in cul-ture, almost all the sphingomyelin and phosphatidylcholine, both of which form less fluid bilayers, are found in the exo-plasmic leaflet. In contrast, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, which form more fluid bilayers, are preferentially located in the cyrosolic leaflet. Because phosphatidylserine and phosphatidylinositol carry a net negative charge, the stretch of amino acids on the cytoplasmic face of a single-pass membrane protein, 111 close proximity to the transmembrane segment, is often enriched in positively charged (Lys, Arg) residues, the "inside posi-tive" rule. This segrt-gation of lipids across the bilayer may influence membrane curvature (see Figure J 0-llc). Unlike particular phospholipids, cholesterol is relatively evenly dis-tributed in both leaflets of cellular membranes. The relative abundance of a particular phospholipid in the two leaflets of a plasma membrane can be determined experimentally on the basis of the susceptibility of phospholipids to hydrolysis


Polar head group ~ r.::l ?~~

-o-P=O f'----.0 CH2 0 I II

?H-O-~C-(CH2lnCH3 CH2 I

(;] b=o5] I (CH2) I n CH3

FIGURE 10-12 Specificity of phospholipases. Each type of phospholipase cleaves one of the susceptible bonds shown in red. The glycerol carbon atoms are indicated by small numbers. In intact cells, only phospholipids in the exoplasmic leaflet of the plasma membrane are cleaved by phospholipases in the surrounding medium. Phospholi-pase C, a cytosolic enzyme, cleaves certain phospholipids in the cytosolic leaflet of the plasma membrane.

by phospholipases, enzymes that cleave the ester bonds via which acyl chains and head groups are connected to the lipid molecule (Figure 10-12). When added to the external me-dium, phospholipases cannot cross the membrane, and thus they cleave off the head groups of only those lipids present in the exoplasmic face; phospholipids in the cytosolic leaflet are resistant to hydrolysis because the enzymes cannot pen-etrate to the cytosolic face of the plasma membrane.

How the asymmetric distribution of phospholipids in membrane leafl.ets arises is still unclear. As noted, in pure bilayers phospholipids do not spontaneously migrate, or flip-flop, from one leaflet to the other. In part, the asym-metry in phospholipid distribution may reflect where these lipids are synthesized in the endoplasmic reticulum and Golgi. Sphingomyelin is synthesized on the luminal (exo-plasmic) face of the Golgi, which becomes the exoplasmic face of the plasma membrane. In contrast, phosphoglycer-Jdes are synthesized on the cyrosolic face of the ER mem-brane, which is topologically equivalent to the cytosolic face of the plasma membrane (see Figure 1 0-5). Clearly, however, this explanation does not account for the prefer-ential location of phospharidylcholine (a phosphoglyceride) in the exoplasmic leaflet. Movement of this phosphoglycer-ide, and perhaps others, from one leaflet to the other in some natural membranes is most likely catalyzed by ATP-powered transport proteins called flippases, which are dis-cussed in Chapter 11.

The preferential location of lipids on one face of the bi-layer is necessary for a variety of membrane-based functions. For example, the head groups of all phosphorylated forms of phosphatidylinositol (see Figure 10-8; PI), an important source of second messengers, face the cytosol. Stimulation of many cell-surface receptors by their corresponding ligand


results in activation of the cytosolic enzyme phospholipase C, which can then hydrolyze the bond connecting the phos-phoinositols to the diacylglycerol. As we will see in Chapter 15, both water-soluble phosphoinositols and membrane-embedded diacylglycerol participate in intracellular signaling pathways that affect many aspects of cell ular metabolism. Phosphatidylserine also is normally most abundant in the cy-tosolic leaflet of the plasma membrane. In the initial stages of platelet stimulation by serum, phosphatidylsenne 1s briefly translocated ro the exoplasmic face, presumably by a flippase enzyme, where it activates enzymes participating in blood clotting. When cells d ie, lipid asymmetry is no longer main-tained, and phosphatidylserine, normally enriched in the cy-rosolic leaflet, is increasingly found in the exoplasmic one. This increased exposure is detected by use of a labeled ver-sion of Annexin V, a protein that specifically binds to phos-phatidylserine, to measure the ooset of programmed cell death (apoptosis).

Cholesterol and Sphingolipids Cluster with Specific Proteins in Membrane Microdomains Membrane lipids are not randomly distributed (evenly mixed) in each leaflet of a bi layer. One hint that lipids may be orga-nized within the leaflets was the discovery that the lipids re-maining after the extraction (solubilization) of plasma membranes with nonionic detergents such as Triton-XlOO predominantly contain two species: cholesterol and sphingo-myelin. Because these two lipids are found in more ordered, less fluid bilayers, researchers hypothesized that they form microdomains, termed lipid rafts, surrounded by other, more fluid phospholipids that are more readily extracted by non-ionic detergents. (We discuss more fully the role of ionic and non ionic detergents in extracting membrane proteins in Section 1 0.2.)

Some biochemical and microscopic evidence supports the existence of lipid rafts, which in natural membranes are typi-cally 50 nm in diameter. Rafts can be disrupted by methyl-~cyclodextrin, wh ich specifically extracts cholesterol out of membranes, or by antibiotics, such as filipin, that sequester cholesterol into aggregates within the membrane. Such find-ings indicate the importance of cholesterol in maintaining the integrity of these rafts. These raft fractions, defined by their insolubility in non ionic detergents, contain a subset of plasma membrane proteins, many of which arc implicated in sensing extracellular signals and transmitting them into the cytosol. Because raft fractions arc enriched in glycolipids, an impor-tant tool for microscopic visualization of raft-type structures in intact cells is the use of fluoresccntly labeled cholera toxin, a protein that specifically binds to certain gangliosides. By bringing many key proteins into dose proximity and stabiliz-ing their interactions, lipid rafts may facilitate signaling by cell-surface receptors and the subsequent activation of cyro-solic events. However, much remains to be learned about the structure and biologica l function of lipid rafts.

.

.

Cells Store Excess Lipids in Lipid Droplets Lipid droplets are vesicular structures, composed of triglycer-ides and cholesterol esters, that originate from the ER and serve a lipid-storage function. When a cell's supply of lipids exceeds the immediate need for membrane construction, ex-cess lipids are relegated to these lipid droplets, readily visual-ized in living cells by staining with a lipophilic dye such as Congo red. Feeding cells with oleic acid, a type of fatty acid, enhances lipid droplet formation. Lipid droplets are not only storage compartments for triglycerides and cholesterol esters, but may also serve as platforms for storage of proteins targeted for degradation. The biogenesis of lipid droplets starts with delamination of the lipid bilayer of the ER, through insertion of triglycerides and cholesterol esters (Figure 10-lJ). The lipid "lens" continues to grow by insertion of more lipid, until fi-nally a lipid droplet is hatched by scission from the ER. The resulting cytoplasmic droplet is thereby enwrapped by a phos-pholipid monolayer. The details of lipid droplet biogenesis as well as their functions remain to be defined more clearly.

ER membrane

fiffr&m~~~ ~~Cholesterol and

triglycerides

"Lens"

Lipid droplet formed from cytoplasmic leaflet

FIGURE 10-13 lipid droplets form by budding and scission from the ER membrane. Lipid droplet formntion begins with the accumula-tion of cholesterol esters and triglycerides (yellow) within the hydro-phobic core of the lipid bilayer. The resulting delamination of the two lipid monolayers causes a "lens" to form, the further growth of which creates a spherical droplet that is then released by scission at the neck. The newly formed droplet is surrounded by a lipid monolayer, derived from the cytosolicleaflet of the ER membrane.

KEY CONCEPTS of Section 1 0.1 The Lipid Bilayer: Composition and Structural Organization Membranes are crucial to cell structure and function. The eukaryotic cell is demarcated from the external environment by the plasma membrane and organized into membrane-limited internal compartments (organelles and vesicles). The phospholipid bilayer, the basic structural unit of all biomembranes, is a two-dimensional lipid sheet with hydro-philic faces and a hydrophobic core, which is impermeable to water-soluble molecules and ions; proteins embedded in the bilayer endow the membrane with specific functions (see Figure 10-1). The primary lipid components of biomembranes are phos-phoglyceridcs, sphingolipids, and sterols such as cholesterol (see Figure 10-8). The term "phospholipid" applies to any amphipathic lipid molecule with a fatty acyl hydrocarbon tail and a phosphate-based polar head group. Phospholipids spontaneously form bilayers and scaled com-partments surrounding an aqueous space (see Figure 10-3). As bilayers, all membranes have an internal (cytosolic) face and an external (exoplasmic) face (see Figure 10-5). Some organelles are surrounded by two, rather than one, membrane bilayer. Most lipids and many proteins are laterally mobile in bio-membranes (see Figure 10-1 0). Membranes can undergo phase transitions from fluid- to gel-like states depending on the tem-perature and composition of the membrane (see Figure 10-9). Different cellular membranes vary in lipid composition (see Table 10-1). Phospholipids and sphingolipids are asymmetri-cally distributed in the two leaflets of the bilayer, whereas cholesterol is fairly evenly distributed in both leaflets. Natural biomembranes generally have a viscous consis-tency with fluidlike properties. In general, membrane fluidity is decreased by sphingolipids and cholesterol and increased hy phosphoglycerides. The lipid composition of a membrane also influences its thickness and curvature (see Figure 10-11 ). Lipid rafts are microdomains containing cholesterol, sphingolipids, and certain membrane proteins that form in the plane of the bilayer. These lipid-protein aggregates might facilitate signaling by certain plasma membrane receptors. Lipid droplets are storage vesicles for lipids, originating in the ER (see Figure 10-13).

10.2 Membrane Proteins: Structure and Basic Functions Membrane proteins are defined by their location within or at the surface of a phospholipid bilayer. Although every bio-logical membrane has the same basic bilayer structure, the

10.2 Membrane Proteins: Structure and Basic Functions 455

proteins associated with a particular membrane are respon-sible for its distinctive activities. The kinds and amounts of proteins associated with biomembranes vary depending on cell type and subcellular location. For example, the inner mi-tochondrial membrane is 76 percent protein; the myelin membrane that surrounds nerve axons, only 18 percent. The high phospholipid content of myelin allows it to electrically insulate the nerve from its environment, as we discuss in Chapter 22. The importance of membrane proteins is evJdent from the finding that approximately a third of all yeast genes encode a membrane protein. The relative abundance of genes for membrane proteins is greater in multicellular organisms, in which membrane proteins have additional functions in cell adhesion.

The lip1d bilayer presents a distinctive two-dimensional hy-drophobic environment for membrane proteins. Some proteins contain segments that are embedded within the hydrophobic core of the phospholipid bilayer; other proteins are associated with the exoplasmic or cytosolic leaflet of the bilayer. Protein domains on the extracellular surface of the plasma membrane generally bind to extracellular molecules, including external signaling proteins, ions, and small metabolites (e.g., glucose, fatty acids), as well as proteins on other cells or in the external environment. Segments of proteins within the plasma mem-brane perform multiple functions, such as forming the chan-nels and pores through which molecules and ions move into and out of cells. Intramembrane segments also serve to orga-nize multiple membrane proteins into larger assemblies within the plane of the membrane. Domains lying along the cytosolic face of the plasma membrane have a wide range of functions, from anchoring cytoskeletal proteins to the membrane to trig-gering intracellular signaling pathways.

In many cases, the function of a membrane protein and the topology of its polypeptide chain in the membrane can be pre-dicted on the basis of its similarity with other well-characterized proteins. In this section, we examine the characteristic struc-tural features of membrane proteins and some of their basic functions. We will describe the structures of several proteins to help you get a feel for the way membrane proteins interact with membranes. More complete characterization of the properties of various types of membrane proteins is presented in later chapters that focus on their structures and activities in the context of their cellular functions.

Proteins Interact with Membranes in Three Different Ways Membrane proteins can be classified into three categories-integral, lipid-anchored, and peripheral-{)n the basis of their position with respect to the membrane (see Figure 10-1). Integral membrane proteins, also called transmembrane pro-tems, span a phospholipid bilayer and comprise three segments. The cytosolic and exoplasmic domains have hydro-philic exterior surfaces that interact with the aqueous envi-ronment on the cytosolic and exoplasmic faces of the membrane. These domains resemble segments of other water-


soluble proteins in their amino acid composition and struc-ture. In contrast, the membrane-spanning segments usually contain many hydrophobic amino acids whose side chains protrude outward and interact with the hydrophobic hydro-carbon core of the phospholipid bilayer. In all transmem-brane proteins examined to date, the membrane-spanning domains consist of one or more a helices or of multiple f3 strands. We discussed the ribosomal synthesis and post-translational processing of soluble cytosolic proteins in Chapters 4 and 8; the process by which integral membrane proteins arc inserted into membranes as part of their synthe-sis is discussed in Chapter 13.

Lipid-anchored membrane proteins are bound covalently to one or more lipid molecules. The hydrophobic segment of the attached lipid is embedded in one leaflet of the mem-brane and anchors the protein to the membrane. The poly-peptide chain itself docs not enter 'the phospholipid bilayer.

Peripheral membrane proteins do not directly contact the hydrophobic core of the phospholipid bilayer. Instead they are bound to the membrane either indirectly by interactions with integral or lipid-anchored membrane proteins or di-rectly by interactions with lipid head groups. Peripheral pro-teins can be bound to either the cytosolic or the exoplasmic face of the plasma membrane. In addition to these proteins, which are closely associated with the bilayer, cytoskeletal filaments can be more loosely associated with the cytosolic face, usually through one or more peripheral (adapter) pro-teins. Such associations with the cytoskeleton provide sup-port for various cellular membranes, helping to determine cell shape and mechanical properties, and play a role in the two-way communication between the cell interior and the exterior, as we learn in Chapter 17. Finally, peripheral pro-teins on the outer surface of the plasma membrane and the exoplasmic domains of integral membrane proteins are often attached to components of the extracellular matrix or to the cell wall surrounding bacterial and plant cells, providing a crucial interface between the cell and its environment.

Most Transmembrane Proteins Have Membrane-Spanning a Helices Soluble proteins exhibit hundreds of distinct localized folded structures, or motifs (see Figure 3-9). In comparison, the reper-toire of folded structures in the transmembrane domains of integral membrane proteins is quite limited, with the hydro-phobic a helix predominating. Proteins containing membrane-spanning a-helical domains are stably embedded in membranes because of energetically favorable hydrophobic and van der Waals interactions of the hydrophobic side chains in the do-main with specific lipids and probably also by ionic interac-tions witlt the polar head groups of the phospholipids.

A single a-helical domain is sufficient to incorporate an integral membrane protein into a membrane. However, many proteins have more than one transmembrane a helix. Typically, a membrane-embedded a helix is composed of a continuous segment of 20-25 hydrophobic (uncharged )

.

(a) Glycophorin A dimer

Cytosolic domain

FIGURE 10- 14 Structure of glycophorin A, a typical single-pass transmembrane protein. (a) Diagram of dime ric glycophorin showing major sequence features and its relation to the membrane. The single 23-residue membrane-spanning a helix in each monomer is composed of amino acids with hydrophobic (uncharged) side chains (red and green spheres). By binding negatively charged phospholipid head groups, the positively charged arginine and lysine residues (blue spheres) near the cytosolic side of the helix help anchor glycophorin in the membrane. Both the extracellular and the cytosolic domains are rich in charged residues and polar uncharged residues; the extracellular

amino acids (see Figure 2-14). The predicted length of such an a helix (3.75 nm) is just sufficient to span the hydrocar-bon core of a phospholipid bilayer. In many membrane pro-teins, these helices are perpendicular to the plane of the membrane, whereas in others, the helices traverse the mem-brane at an oblique angle. The hydrophobic side chains pro-trude outward from the helix and form van der Waals interactions with the farcy acyl chains in the bilayer. In con-trast, the hydrophilic amide peptide bonds are in the interior of the a helix (see Figure 3-4); each carbonyl (C=O) group forms a hydrogen bond with the amide hydrogen atom of the amino acid four residues toward the C-terminus of the helix. These polar groups are shielded from the hydrophobic interior of the membrane.

To help you get a better sense of the structures of proteins with a-helical domains, we will briefly discuss four different kinds of such protems: glycophorin A, G protein-coupled receptors, aquaporins (water/glycerol channels), and T-cell receptor for antigen.

Glycophorin A, the major protein in the erythrocyte plasma membrane, is a representative single-pass transmem-

(b) Transmembrane coiled-coil domain

t:w.:;->~---Coiled-coil dimer stabilized by van der Waals interactions between adjacent side chains

domain is heavily glycosylated, with the carbohydrate side chains (green diamonds) attached to specific serine, threo'nine, and aspara-gine residues. (b) Molecular model of the transmembrane domain of dime ric glycophorin corresponding to residues 73-96. The hydrophobic side chains of the a helix in one monomer are shown in pink; those in the other monomer, in green. Residues depicted as space-filling structures participate in van der Waals interactions that stabilize the coiled-coil dimer. Note how the hydrophobic side chains project outward from the helix, toward what would be the surrounding fatty acyl chains. [Part (b) adapted from K. R. MacKenzie et al., 1997, Science 276:131.]

brane protein, which contains only one membrane-spanning a helix (Figure 10-14 ). The 23-residue membrane-spanning a helix is composed of amino acids with hydrophobic (un-charged) side chains, which interact with fatty acyl chains in the surrounding bilayer. In cells, glycophorin A typically forms dimers: the transmembrane helix of one glycophorin A polypeptide associates with the corresponding transmem-brane helix in a second glycophorin A to form a coiled-coil structure (Figure 10-14b). Such interactions of membrane-spanning a helices are a common mechanism for creating dtmeric membrane proteins, and many membrane proteins form oligomers (two or more polypeptides bound together noncovalenrly) by interactions between their membrane-spanning helices.

A large and important group of integral proteins is de-fined by the presence of seven membrane-spanning a helices; this includes the large family of G protein-

(a) Bacteriorhodopsin

Exterior

Membrane

Cytosol

(b) Glycerol channel Back

FIGURE 10-1 5 Structural models of two multipass membrane proteins. (a) Bacteriorhodopsin, a photoreceptor in certain bacteria. The seven hydrophobic a helices in bacteriorhodopsin traverse the lipid bilayer roughly perpendicu lar to the plane of the membrane. A retinal molecule (black) covalently attached to one helix absorbs light. The large class of G protein-coupled receptors in eukaryotic cells also has seven membrane-spanning a helices; their t hree-dimensional structure is thought to be similar to that of bacteriorhodopsin. (b) Two views of the glycerol channel Glpf, rotated 180 with respect to each other along an axis perpendicular to the plane of the membrane. Note

illustrates the general structure of all these proteins (Figure 1 0-15a). Absorption of light by the retinal group covalently attached to this protein causes a conformational change in the protein that results in the pumping of protons from the cytosol across the bacterial membrane to the extracellular space. The proton concentration gradient thus generated across the membrane is used to synthesize ATP during pho-tosynthesis (Chapter 12). In the high-resolution structure of bacteriorhodopsin the positions of all the individual amino acids, retinal, and the surrounding lipids are clearly defined. As might be expected, virtually all of the amino acids on the exterior of the membrane-spanning segments of bacteriorho-dopsin are hydrophobic, permitting energetically favorable interactions with the hydrocarbon core of the surrounding lipid bilayer.

The aquaporins are a large family of highly conserved proteins that transport water, glycerol, and other hydro-philic molecules across biomembranes. They illustrate sev-eral aspects of the structure of multipass transmembrane proteins. Aquaporins arc tc tramers of four identical sub-units. Each of the four subunits has six membrane-spanning a helixes, some of which traverse the membrane at oblique angles rather than perpendicularly. Because the aquaporins have similar structures, we will focus on one, the glycerol channel Glpf, that has an especially well-defined structure determined by x-ray diffraction studies (Figure 10-15b). This


Front Half elices

several membrane-spanning a helices that are at oblique angles, the two helices that penetrate only halfway through the membrane (purple with yellow arrows), and one long membrane-spanning helix with a "break" or distortion in the middle (purple with yellow line). The glycerol molecule in the hydrophilic "core" is colored red. The st ructure was approximately positioned in the hydrocarbon core ofthe membrane by finding the most hydrophobic 3-~m slab of the protein perpendicular to the membrane plane. [Part (a) after H. Luecke et at., 1999, J. Mol. Bioi. 291:899. Part (b) after J. Bowie, 2005, Nature 438:581-589, and D. Fu et at., 2000, Science 290:481-486.]

aquaporin has one long transmembrane helix with a bend in the middle, and more strikingly, there are two a helices that penetrate only halfway through the membrane. TheN-termini of these helices face each other (yellow N's in the figure), and together they span the membrane at an oblique angle. Thus some membrane-embedded helices- and other, nonhe-lical, structures we will encounter later-do not traverse the entire bi layer. As we will see in Chapter 11, these short heli -ces in aquapor ins form part of the glycerol/water-selective pore in the middle of each subunit. This highlights the con-siderable diversity in the ways membrane-spanning a helices interact with the lipid bilayer and with other segments of the protein.

The specificity of phospholipid-protein interactions is evi-dent from the structure of a different aquaporin, aquaporin 0 (Figure 1 0-16). Aquaporin 0 is the most abundant protein in the plasma membrane of the fiber cells that make up the bulk of the lens of the mammalian eye. Like other aquaporins, it is a tetramer of identical subunits. The protein's surface is not t-uvered by a set of uniform bind ing sites for phospholipid molecules. Instead, fatty acyl side chains pack tightly against the irregular hydrophobic outer surface of the protein; these lipids are referred to as annular phospholipids, because they from a tight ring (annulus) of lipids that exchange less easily with bulk phospholipids in the bilayer. Some of the fatty acyl chains are straight, in the all-trans conformation (Chapter 2),

.

'

@ PODCAST: Annular Phospholipids

Exterior

Membrane

Interior

whereas others are kinked in order ro interact with bulky hydroph il ic side chains on the surface of the protein. Some of the li pid head groups are parallel to the su rface of the membrane, as is the case in purified phospholipid bilayers. Others, however, are oriented almost at right angles to the plane of the membrane. Thus there can be specific interac-tions between phospholipids and membrane-spanning pro-teins, and the function of many membrane proteins can be

ER lumen

C030E

Cytosol

ER lumen

~ 1:

Membrane I

'-r

Cytosol

FIGURE 10-16 Annular phospholipids. Side view of the three-dimensional structure of one subunit of the lens-specific aquaporin 0 homotetramer, crystal lized in the presence of the phospholipid dimyristoylphosphatidylcholine, a phospholipid with 14 carbon-saturated fatty acyl chains. Note the lipid molecules forming a bilayer shell around the protein. The protein is shown as a surface plot (the liqhter background molecule). The lipid molecules are shown in space-fill format; the polar lipid head groups (grey and red) and the lipid fatty acyl chains (black and grey) form a bilayer with almost uniform thickness around the protein. Presumably, in the membrane, lipid fatty acyl chains will cover the whole of the hydrophobic surface of the protein; only the most ordered of the lipid molecules will be resolved in the crystallographic structure. [After A. Lee, 2005, Nature 438:569-570, and T. Gonen et al., 2005, Nature 438:633-688.)

affected by the specific types of phospholipid present in the bilayer.

In addition to the predominantly hydrophobic (un-charged) residues that serve to embed int~gral membrane proteins in the bi layer, many such a-helical transmembrane segments do contain polar and/or charged residues. Their amino acid side chains can be used to guide the assembly and stabilization of multimeric membrane proteins. The T-ccll re-ceptor for antigen is a case in point: it is composed of four separate dimers, the interactions of which are driven by charge-charge interactions between a helices at the appropri-ate "depth" in the hydrocarbon core of the lipid bi layer (Fig-ure 10- 17). The electrostatic attraction of positive and negative charges on each dimer helps the dimers to "find each other." T hus charged residues in otherwise hydrophobic transmembrane segments can help guide assembly of multi-meric membrane proteins.

FIGURE 10-17 Charged residues can orchestrate assembly of multimeric membrane proteins. The T-cell receptor (TCR) for antigen is composed of four separate dimers: an al3 pair directly responsible for antigen recognition, and accessory subunits collectively referred to as the CD3 complex. These accessories include the-y, o, E:, and~ subunits. The~ subunits form a disulfide-linked homodimer. The "Y and 8 subunits occur in complex with an E subunit, to generate a "YE and a 1iE pair. The transmembrane segments of the TCR a and 13 chains each contain positively charged residues (blue). These allow recruitment of corresponding 8E: and 'fE: heterodimers, which carry negative charges (red) at the appropriate depth in the hydrophobic core of the bilayer. The~ homodimer docks onto the charges in the TCR a chain (dark green), w hile the 'fE: and 8E: subunit pairs find their corresponding partners deeper down in the hydrophobic core on both the TCR a and TCR 13 chain (light green). Charged residues in otherwise nonpolar transmembrane segments can thus guide assembly of higher order structures. [After K.W. Wucherpfennig, et al., 2010, Cold Spring Horb Perspect Bio/, 2.)


Multiple 13 Strands in Porins Form Membrane-Spanning "Barrels" The porins are a class of transmembrane proteins whose struc-ture differs radically from that of other integral proteins based on a-helical transmembrane domains. Several types of porins are found in the outer membrane of gram-negative bacteria such as E. coli and in the outer membranes of mitochondria and chloroplasts. The outer membrane protects an intestinal bacterium from harmful agents (e.g., antibiotics, bile salts, and proteases) but permits the uptake and disposal of small hydro-philic molecules, including nutrients and waste products. Dif-ferent types of porins in the outer membrane of an E. coli cell provide channels for the passage of specific types of disaccha-rides or other small molecules as well as of ions such as phos-phate. The amino acid sequences of porins contain none of the long, continuous hydrophobic segments typical of integral pro-teins with a-helical membrane-spanning domains. Rather, it is the entire outer surface of the fully folded porin that displays its hydrophobic character to the hydrocarbon core of the lipid bilayer. X-ray crystallography shows that porins are trimers of identical subunits. In each subunit, 16 13 strands form a sheet that twists into a barrel-shaped structure with a pore in the center (Figure 10-18). Unlike a typical water-soluble globular

Peri plasm

FIGURE 10-18 Structural model of one subunit of OmpX, a porin found in the outer membrane of E. coli. All porins are trimeric transmembrane proteins. Each subunit is barrel shaped, with 13 strands forming the wall and a transmembrane pore in the center. A band of aliphatic (hydrophobic and noncyclic) side chains (yellow) and a border of aromatic (ring-containing) side chains (red) position the protein in the bilayer. [After G. E. Schulz, 2000, Curr. Opm. Struc. Bioi. 1 0:443.]


protein, a porin has a hydrophilic interior and a hydrophobic exterior; in this sense, porins are inside out. In a porin mono-mer, the ourward-facing side groups on each of the 13 strands are hydrophobic and form a nonpolar ribbonlike band that encircles the outside of the barrel. This hydrophobic band in-teracts with the fatty acyl groups of the membrane lipids or with other porin monomers. The side groups facing the inside of a porin monomer are predominantly hydroph ilic; they line the pore through which small water-soluble molecules cross the membrane. (Note that the aquaporins discussed above, de-spite their name, are not porins and contain multiple trans-membrane a helices.)

Covalently Attached Lipids Anchor Some Proteins to Membranes In eukaryotic cells, covalently attached lipids can anchor some otherwise typically water-soluble proteins to one or the other leaflet of the membrane. In such lipid-anchored proteins, the lipid hydrocarbon chains are embedded in the bilayer, but the protein itself does not enter the bilayer. The lipid anchors used to anchor proteins to the cytosolic face are not used for the exoplasmic face and vice versa. .

One group of cytosolic proteins are anchored to the cyto-solic face of a membrane by a fatty acyl group (e.g., myristate or palmitate) covalently attached to an N-terminal glycine residue, a process called acylation (Figure 1 0-19a ). Retention of such proteins at the membrane by the N-terminal acyl an-chor may play an important role in a membrane-associated function. For example, v-Src, a mutant form of a cellular tyro-sine kinase, induces abnormal cellular growth that can lead to cancer but does so only when it has a myristylated N-terminus.

A second group of cytosolic proteins are anchored to membranes by a hydrocarbon chain attached to a cysteine residue at or near the C-terminus, a process called prenyl-ation (Figure 10-19b). Prcnyl anchors are built from 5-carbon isoprene units, which, as detailed in the following section, are also used in the synthesis of cholesterol. In prenylation, a 15-carbon farnesyl or 20-carbon geranylgeranyl g roup is bound through a thioether bond to the -SH group of a C-terminal cysteine residue of the protein. In some cases, a sec-ond geranylgeranyl group or a fatty acyl palmitate group is linked to a nearby cysteine residue. The additional hydrocar-bon anchor is thought to reinforce the attachment of the protein to the membrane. For example, Ras, a GTPase su-perfamily protein that functions in intracellular signaling (Chapter 15), is recruited to the cytosolic face of the plasma membrane by such a double anchor. Rab proteins, which also belong to the GTPase superfamily, are similarly bound to the cytosolic surface of intracellular vesicles by prenyl an-chors; these proteins arc requireu fur the fusion of vesicles with their target membranes (Chapter 14).

Some cell-surface proteins and specialized proteins with distinctive covalently attached polysaccharides called pro-teoglycans (Chapter 20 ) are bound to the exoplasmic face of the plasma membrane by a third type of anchor group, glycosylphosphatidylinositol (GPI). The exact structures of

. '

(c) GPI anchor Exterior

Cytosol

(a) Acylation (b) Prenylation FIGURE 10-19 Anchoring of plasma-membrane proteins to the bilayer by covalently linked hydrocarbon groups. (a) Cytosolic proteins such as v-Src are associated with the plasma membrane through a single fatty acyl chain attached to the N-terminal glycine (Giy) residue of the polypeptide. Myristate ((14) and palmitate (C 16) are common acyl anchors. (b) Other cytosolic proteins (e.g., Ras and Rab proteins) are anchored to the membrane by prenylation of one or two cysteine (Cys) residues, at or near the (-terminus. The anchors are farnesyl ((15) and geranylgeranyl (C20) groups, both of which are unsaturated. (c) The lipid anchor on the exoplasmic surface of the plasma membrane is glycosylphosphatidylinositol (GPI). The phosphatidylinosi-tol part (red) of this anchor contains two fatty acyl chains that extend into the bilayer. The phosphoethanolamine unit (purple) in the anchor links it to the protein. The two green hexagons represent sugar units, which vary in number, nature, and arrangement in different GPI anchors. The complete structure of a yeast GPI anchor is shown in Figure 13-15. [Adapted from H. Sprong et al., 2001, Nature Rev. Mol. Cell Bioi. 2:504.]

G Pi anchors vary greatly in different cell types, but they always contain phosphatidylinositol (PI), whose two fatty acyl chains extend into the lipid bilayer just like those of typical membrane phospholipids; phosphoethanolamine, which covalently links the anchor to the C-terminus of a protein; and several sugar residues (Figure 10-19c). Therefore GPI anchors are glycolip-ids. The GPI anchor is both necessary and sufficient for binding proteins co the membrane. for instance, treatment of cells with phospholipase C, which cleaves the phosphate-glycerol bond in phospholipids and in GPI anchors (see Figure 10-12), releases GPI-anchored proteins such as Thy-1 and placental alkaline phosphatase (PLAP) from the cell surface.

All Transmembrane Proteins and Glycolipids Are Asymmetrically Oriented in the Bilayer Every type of transmembrane protein has a specific orienta-tion, known as its topology, with respect to the membrane faces. Its cytosolic segments are alway!> facing the cytoplasm,

and exoplasmic segments always face the opposite side of the membrane. This asymmetry in protein orientation confers different properties on the two membrane faces. The orienta-tion of different types of transmembrane proteins is estab-lished during their synthesis, as we describe in Chapter 13. Membrane proteins have never been observed to flip-flop across a membrane; such movement, requiring a transient movement of hydrophilic amino acid residues through the hydrophobic interior of the membrane, would be energeti-cally unfavorable. Accordingly, the asymmetric topology of a transmembrane protein, which is established during its bin-synthetic insertion into a membrane, is maintained through-out the protein's lifetime. As Figure 10-6 shows, membrane proteins retain their asymmetric orientation in the membrane during membrane budding and fusion events; the same seg-ment always faces the cytosol and the same segment is always exposed to the exoplasmic face. In proteins with multiple transmembrane segments (multipass or polytopic membrane proteins), orientation of individual transmembrane segments can be affected by changes in phospholipid composition.

Many transmembrane proteins contain carbohydrate chains covalently linked to serine, threonine, or asparagine side chains of the polypeptide. Such transmembrane glycoproteins are always oriented so that all carbohydrate chains are in the exoplasmic domain (see Figure 10-14 for the example of gly-cophorin A). Likewise, glycolipids, in which a carbohydrate chain is attached to the glycerol or sphingosine backbone of a membrane lipid, are always located in the exoplasmic leaflet with the carbohydrate chain protruding from the membrane surface. The biosynthetic basis for the asymmetric glycosyl-ation of proteins is described in Chapter 14. Both glycopro-teins and glycolipids are especially abundant in the plasma membranes of eukaryotic cells and in the membranes of the intracellular compartments that establish the secretory and en-docytic pathways; they are absent from the inner mitochon-drial membrane, chloroplast lamellae, and several other intracellular membranes. Because the carbohydrate chains of glycoproteins and glycolipids in the plasma membrane extend into the extracellular space, they are available to interact with components of the extracellular matrix as well as lectins (pro-teins that bind specific sugars), growth factors, and antibodies.

One important consequence of such interactions is il-lustrated by the A, B, and 0 blood group antigens.

These three structurally related oligosaccharide components of certain glycoproteins and glycolipids are expressed on the surfaces of human red blood cells and many other cell types (Figure 10-20). All humans have the enzymes for synthesizing 0 antigen. Persons with type A blood also have a glycosyl-transferase enzyme that adds an extra modified monosaccha-ride called N-acetylgalactosamine to 0 antigen to form A antigen. Those with type B blood have a different transferase that adds an extra galactose to 0 antigen to form B antigen. People with both transferases produce both A and B antigen (AB blood type); those who lack these transferases produce 0 antigen only (0 blood type).


Lipid or protein

Lipid or protein

Lipid or protein

GaiNAc 1 transferase

Glc = Glucose Gal = Galactose GlcNAc = N-Acetylglucosamine GaiNAc = N-Acetylgalactosamine Fuc =Fucose

A antigen

0 antigen

8 antigen

FIGURE 10-20 Human ABO blood group antigens. These antigens are oligosaccharide chains covalently attached to glycolipids or glycoproteins in the plasma membrane. The terminal oligosaccharide sugars distinguish the three antigens. The presence or absence of the glycosyltransferases that add galactose (Gal) or N-acetylgalactosamine (GaiNAc) to 0 antigen determine a person's blood type.

People whose erythrocytes lack the A antigen, the B anti-gen, or both on their surface normally have antibodies against the missing antigen(s) in their serum. Thus if a type A or 0 person receives a transfusion of type B blood, antibodies against the B antigen will bind to the introduced red cells and trigger their destruction. To prevent such harmful reactions, blood group typi~g and appropriate matching of blood donors and recipients are required in all transfusions (Table 10-2).

Lipid-Binding Motifs Help Target Peripheral Proteins to the Membrane Many water-soluble enzymes use phospholipids as their sub-strates and thus must bind to membrane surfaces. Phospho-

TABLE 10-2 ABO Blood Groups

Blood Group Antigens on RBCs*

A A

B B

AB A and B

0 0

sec Figure 10-20 for anrigen structures.


lipases, for example, hydrolyze various bonds in the head groups of phospholipids (see Figure 10-12), and thereby play a variety of roles in cells-helping to degrade damaged or aged cell membranes, generating precursors to signaling molecules, and even serving as the active components in many snake venoms. Many such enzymes, including phos-pholipases, initially bind to the polar head groups of mem-brane phospholipids to carry out their catalytic functions. The mechanism of action uf phospholipase A2 illustrates how such water-soluble enzymes can reversibly interact with membranes and catalyze reactions at the interface of an aqueous solution and lipid surface. When this enzyme is in aqueous solution, its Ca2 -containing active site is buried in a channel lined with hydrophobic amino acids. The enzyme binds with greatest affinity to bilayers composed of nega-tively charged phospholipids (e.g., phosphotidylserine). This finding suggests that the rim of positively charged lysine and arginine residues around the entrance to the catalytic chan-nel is particularly important in binding (Figure 10-21 a). Binding induces a conformational change in phospholipase A2 that strengthens its binding to the phospholipid heads and opens the hydrophobic channel. As a phospholipid mol-ecule moves from the bilayer into the channel, the enzyme-bound Ca2+ binds to the phosphate in the head group, thereby positioning the ester bond to be cleaved in the cata-lytic site (Figure 10-21b) and so releasing the acyl chain.

Proteins Can Be Removed from Membranes by Detergents or High-Salt Solutions Membrane proteins are often difficult to purify and study, mostly because of their tight association with membrane lip-ids and other membrane proteins. Detergents are amphipa-thic molecules that disrupt membranes by intercalating into phospholipid bilayers and can thus be used to solubilize lip-ids and many membrane proteins. The hydrophobic part of a detergent molecule is attracted to the phospholipid hydro-carbons and mingles with them readily; the hydrophilic part is strongly attracted to water. Some detergents such as the bile salts are natural products, but most are synthetic mole-cules developed for cleaning and for dispersing mixtures of oil and water in the food industry (e.g., creamy peanut butter)

Serum Antibodies Can Receive Blood Types - --

Anti-B A and 0

Ami-A Band 0

None All

Anti-A and anti-B 0

FIGURE 10-21 Lipid-binding surface and mechanism of action of phospholipase A2 (a) A structural model of the enzyme showing the surface that interacts w ith a membrane. This lipid-binding surface contains a rim of positively charged arginine and lysine residues, shown in blue surrounding the cavity of the catalytic active site, in which a substrate lipid (red stick structure) is bound. (b) Diagram of catalysis by phospholipase A2 When docked on a model lipid mem-brane, positively charged residues of the binding site bind to nega-tively charged polar groups at the membrane surface. This binding triggers a small conformational change, opening a channel lined with hydrophobic amino acids that leads from the bilayer to the catalytic site. As a phospholipid moves into the channel, an enzyme-bound Ca2

ion (green) binds to the head group, positioning the ester bond to be cleaved (red) next to the catalytic site. [Part (a) adapted from M. H. Gelb et al., 1999, Curr. Opm. Struc. Bioi. 9:428. Part (b), see D. Blow, 1991, Nature 351 :444.]

IONIC DETERGENTS

H3C L...._ ___ _

(Figure 10-22). Ionic detergents, such as sodium deoxycholate and sodium dodecylsulfate (SDS), contain a charged group; nonionic detergents, such as Triton X-100 and octylglucoside, lack a char

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