All biological membranes share certain fundamental properties. They are impermeable to most polar or charged solutes, but permeable to nonpolar compounds; are 5 to 8 nm thick; appear trilaminar (threelayered) when viewed in cross section with the electron microscope (see Fig. 10-1). The combined evidence from electron microscopy, chemical composition, and physical studies of permeability and of the motion ofindividual protein and lipid molecules within membranes supports the fluid mosaic model for the structure of biological membranes (Fig. 10-4). Amphipathic phospholipids and sterols form a lipid bilayer, with the nonpolar regions of lipids facing each other at the core of the bilayer and their polar head groups facing outward. In this lipid bilayer, globular proteins are embedded at irregular intervals, held by hydrophobic interactions between the membrane lipids and hydrophobic domains in the proteins. Some proteins protrude from one or the other face of the membrane; most span its entire width. The orientation of proteins in the bilayer is asymmetric, giving the membrane "sidedness"; the protein domains exposed on one side of the bilayer are different from those exposed on the other side, reflecting functional asymmetry. The individual lipid and protein subunits in a membrane form a fluid mosaic; its pattern, unlike a mosaic of ceramic tile and mortar, is free to change constantly. The membrane mosaic is fluid because the interactions among lipids, and between lipids and proteins, are noncovalent, leaving individual lipid and protein molecules free to move laterally in the plane of the membrane.
We will now look at some of these features of the fluid mosaic model in more detail, and consider the experimental evidence that supports it.
We will now look at some of these features of the fluid mosaic model in more detail, and consider the experimental evidence that supports it.
A Lipid Bilayer Is the Basic Structural Element
| We saw in Chapter 9 that lipids, when suspended in water, spontaneously form bilayer structures that are stabilized by hydrophobic interactions (see Fig. 9-14). The thickness of biological membranes (5 to 8 nm, measured by electron microscopy) is about that expected for a lipid bilayer 3 nm thick with proteins protruding on each side. X-ray diffraction by membranes shows the distribution of electron density expected for a bilayer structure. Liposomes (lipid vesicles) formed in the laboratory show the same relative impermeability to polar solutes as is seen in biological membranes (although the latter are permeable to solutes for which they have specific transporters). In short, all evidence indicates that biological membranes are constructed of lipid bilayers. Membrane lipids are asymmetric in their distribution on the two faces of the bilayer, although the asymmetry, unlike that of membrane proteins, is not absolute. In the plasma membrane, for example, certain lipids are typically found primarily in the outer face of the bilayer, and others in the inner (cytoplasmic) face (Fig. 10-5). |
Membrane Lipids Are in Constant Motion
| Although the lipid bilayer structure itself is stable, the individual phospholipid and sterol molecules have great freedom of motion within the plane of the membrane (Fig. 10-6). They diffuse laterally so fast that an individual lipid molecule can circumnavigate an erythrocyte in a few seconds. The interior of the bilayer is also fluid; individual hydrocarbon chains of fatty acids are in constant motion produced by rotation about the carbon-carbon bonds of the long acyl side chains.
The degree of fluidity depends on lipid composition and temperature. At low temperature, relatively little lipid motion occurs and the bilayer exists as a nearly crystalline (paracrystalline) array. Above a temperature that is characteristic for each membrane, lipids can undergo rapid motion. The temperature of the transition from paracrystalline solid to fluid depends upon the lipid composition of the membrane. Saturated fatty acids pack well into a paracrystalline array, but the kinks in unsaturated fatty acids (see Fig. 9-1) interfere with this packing, preventing the formation of a paracrystalline solid state. The higher the proportion of saturated fatty acids, the higher is the solid-to-fluid transition temperature of the membrane. The sterol content of a membrane also is an important determinant of this transition temperature. The rigid planar structure of the steroid nucleus, inserted between fatty acyl side chains, has two effects on fluidity: below the temperature of the solid-to-fluid transition, sterol insertion prevents the highly ordered packing of fatty acyl chains, and thus fluidizes the membrane. Above the thermal transition point, the rigid ring system of the sterol reduces the freedom of neighboring fatty acyl chains to move by rotation about carbon-carbon bonds, and thus reduces the fluidity in the core of the bilayer. Sterols therefore tend to moderate the extremes of solidity and fluidity of the membranes that contain them. Both microorganisms and cultured animal cells regulate their lipid composition so as to achieve a constant fluidity under various growth conditions. For example, when cultured at low temperatures, bacteria synthesize more unsaturated fatty acids and fewer saturated ones than when cultured at higher temperatures (Table 10-3). As a result of this adjustment in lipid composition, membranes of bacteria cultured at high or low temperature have about the same degree of fluidity. |
Membrane Proteins Penetrate and Span the Lipid Bilayer
| When individual protein molecules and multiprotein complexes in freeze-fractured biological membranes are visualized with the electron microscope (Box 10-1), some proteins appear on only one face of the membrane; most span the full thickness of the bilayer, and protrude from both inner and outer membrane surfaces. Among the latter are some proteins that conduct solutes or signals across the membrane. Membrane protein localization has also been investigated with reagents that react with protein side chains but cannot cross membranes (Fig. 10-7). The human erythrocyte is convenient for such studies, because the plasma membrane is the only membrane present. |
Membrane Proteins Are Oriented Asymmetrically
One further fact may be deduced from the results of the experiments with glycophorin: it does not move from one face of the bilayer to the other; its disposition in the membrane is asymmetric. Similar studies of other membrane proteins show that each has a specific orientation in the bilayer, and that protein reorientation by flip-flop diffusion occurs seldom, if ever. Furthermore, glycoproteins of the plasma membrane are invariably situated with their sugar residues on the outer surface of the cell. As we shall see, the asymmetric arrangement of membrane proteins results in functional asymmetry; all the molecules of a given ion pump, for example, have the same orientation and therefore all pump in the same direction.Integral Membrane Proteins Are Insoluble in Water
Membrane proteins may be divided operationally into two groups: integral (intrinsic) proteins, which are very firmly bound to the membrane, and peripheral (extrinsic) proteins, which are bound more loosely, or reversibly. Peripheral membrane proteins can be released from membranes by relatively mild treatments (Fig. 10-9), and once released from the membrane they are generally water soluble. In contrast, the release of integral proteins from membranes requires the action of agents (detergents, organic solvents, or denaturants) that interfere with hydrophobic interactions. Even after integral proteins have been solubilized, removal of the detergent may cause the protein to precipitate as an insoluble aggregate. The insolubility of integral membrane proteins results from the presence of domains rich in hydrophobic amino acids; hydrophobic interactions between the protein and the lipids of the membrane account for the firm attachment of the protein.
Some Integral Proteins Have Hydrophobic Transmembrane AnchorsIntegral membrane proteins generally have domains rich in hydrophobic amino acids. In some proteins, there is a single hydrophobic sequence in the middle of the protein (as in glycophorin) or at the amino or carboxyl terminus. Other membrane proteins have multiple hydrophobic sequences, each long enough to span the lipid bilayer when in the a-helical conformation. |
One of the best-studied membrane-spanning proteins, bacteriorhodopsin, contains seven very hydrophobic internal sequences, and crosses the lipid bilayer seven times. Bacteriorhodopsin is a lightdriven proton pump that is densely packed in regular arrays in the purple membrane of the bacterium Halobacterium halobium. When these arrays are viewed with the electron microscope from several angles, the resulting images allow a three-dimensional reconstruction of the bacteriorhodopsin molecule (Fig. 10-10). Seven α-helical segments, each traversing the lipid bilayer, are connected by nonhelical loops at the inner or outer face of the membrane. The amino acid sequence of bacteriorhodopsin has seven segments with about 20 hydrophobic residues, each segment just long enough to make a helix that spans the lipid bilayer. Hydrophobic interactions between the nonpolar amino acids and the acyl side chains of the membrane lipids firmly anchor the protein in the membrane, providing a transmembrane pathway for proton translocation
|
The Structure of a Crystalline Integral Membrane Protein Has Been Determined
The same techniques that have allowed determination of the threedimensional structures of many soluble proteins can in principle be applied to membrane proteins. However, very few membrane proteins have been crystallized; they tend instead to form amorphous aggregates. One instructive exception is the photosynthetic reaction center from a purple bacterium (Fig. 10-11). The protein has four subunits, three of which contain α-helical segments that span the membrane. These segments are rich in nonpolar amino acids, and their hydrophobic side chains are oriented toward the outside of the protein, interacting with the hydrocarbon side chains of membrane lipids. The architecture of the reaction center protein is therefore the inverse of that seen in most water-soluble proteins, which have their hydrophobic residues buried within the protein core and their hydrophilic residues on the surface available for polar interactions with water (recall the structures of myoglobin and hemoglobin, for example). The structures of only a few membrane proteins are known, but the hydrophobic exterior of the reaction center protein seems to be typical of integral membrane proteins.Peripheral Proteins Associate Reversibly with the Membrane
Many peripheral proteins are held to the membrane by electrostatic interactions and hydrogen bonding with the hydrophilic domains of integral membrane proteins, and perhaps with the polar head groups of membrane lipids. They can be released by relatively mild treatments that interfere with electrostatic interactions or break hydrogen bonds (see Fig. 10-9). These peripheral proteins may serve as regulators of membrane-bound enzymes, or as tethers that connect integral membrane proteins to intracellular structures or limit the mobility of certain membrane proteins.Lipids attached covalently to certain membrane proteins (see Fig. 10-3) anchor these proteins to the lipid bilayer by hydrophobic interactions. Proteins thus held can be released from the membrane by the breakage of a single bond; the action of phospholipase C or D, for example, frees the membrane protein from the hydrophobic portion of a phosphatidylinositol "anchor" (see Fig. 10-9). It seems likely that this type of quick-release mechanism gives cells the capacity to change their membrane surface architecture rapidly, or to alter the subcellular localization of proteins that shuttle between membrane and cytosol.
.Although these proteins with lipid anchors resemble integral membrane proteins in that they can be solubilized by detergent treatment, they are generally considered peripheral membrane proteins on the basis of their other properties: their association with the membrane is often weak and reversible, they do not contain long hydrophobic sequences, and once solubilized (by phospholipase action, for example), they behave like typical soluble proteins.
Membrane Proteins Diff-use Laterally in the BilayerMany membrane proteins behave as though they were afloat in a sea of lipids. We noted earlier that membrane lipids are free to diffuse laterally in the plane of the bilayer, and are in constant motion. The experiment diagrammed in Figure 10-12 shows that this is also true of some membrane proteins. Other experimental techniques confirm that many but not all membrane proteins undergo rapid lateral diffusion, but there are many exceptions to this generalization. Some membrane proteins associate with adjacent membrane proteins to form large aggregates ("patches") on the surface of a cell or organelle, in which individual protein molecules do not move relative to one another. Acetylcholine receptors (p. 292) form dense patches at synapses. Other membrane proteins are anchored to internal structures that prevent their free diffusion in the membrane bilayer. In the erythrocyte membrane, both glycophorin and the chloride-bicarbonate exchanger (p. 286) are tethered from the inside to a filamentous cytoskeletal protein, spectrin (Fig. 10-13). |
Membrane Fusion Is Central to Many Biological Processes
| Although membranes are stable, they are by no means static. The fluid mosaic structure is dynamic and flexible enough to allow fusion of two membranes. Within the endomembrane system described in Chapter 2 (see Fig. 2-10) there is constant reorganization of the membranous compartments, as small vesicles bud from the Golgi complex carrying newly synthesized lipids and proteins to other organelles and to the plasma membrane. Exocytosis, endocytosis, fusion of egg and sperm cells, and cell division all involve membrane reorganization in which the fundamental operation is fusion of two membrane segments without loss of continuity (Fig. 10-14 |
To fuse, two membranes must first approach each other within molecular distances (a few nanometers). Much evidence suggests that an increase in intracellular Ca2+ concentration is the signal for certain fusion events such as exocytosis. Annexins are a family of proteins located just beneath the plasma membrane. They bind avidly to the head groups of phospholipids in bilayers, but only in the presence of Ca2+. Some annexins also associate with specific intracellular vesicles fated for exocytosis. These proteins cause clumping of liposomes in vitro, presumably by cross-linking lipid molecules of two different vesicles. In one simple model of membrane fusion (Fig. 10-15), annexins hold two membranes in close and stable apposition in the first step of fusion.
Another family of proteins believed to act in fusion is the fusion proteins, which are typified by the integral membrane protein HA, essential for the entry of the influenza virus into host cells (Fig. 10-14). (Note that these "fusion proteins" are unrelated to the products of two fused genes, also called fusion proteins, discussed in Chapter 28.) Like most other fusion proteins, the H A protein contains two regions rich in nonpolar amino acids, one a typical membrane-spanning domain, the other (the "fusion peptide") rich in Ala and Gly residues. The fusion protein may bridge the two membranes, with its membrane-spanning domain in one membrane and the fusion peptide inserted into the other (Fig. 10-15).
Fusion proteins may bring about transient distortions of the bilayer structure in the region of fusion. Physical studies of pure phospholipids in vitro have shown that several nonbilayer structures, such as inverted micelles, can form under some circumstances. In the model illustrated in Figure 10-15, an alternative structure exists in equilibrium with the bilayer, and represents the transition structure between unfused and fused membranes. Fusion proteins are believed to favor the formation of the transition structure, thus easing the phospholipid reorganization that results in fusion. In addition to annexins and fusion proteins, several, perhaps many, other proteins are probably involved; the machinery for fusion may be much more complex than implied by the simple model described here.
