5:  5.3 Biomembranes: Structural Organization and Basic Functions

Phospholipids Are the Main Lipid Constituents of Most Biomembranes

The most abundant lipid components in most membranes are phospholipids, which are amphipathic molecules (i.e., they have a hydrophilic and a hydrophobic part). In phosphoglycerides, a principal class of phospholipids, fatty acyl side chains are esterified to two of the three hydroxyl groups in glycerol, and the third hydroxyl group is esterified to phosphate. The phosphate group is also esterified to a hydroxyl group on another hydrophilic compound, such as choline in phosphatidylcholine (Figure 5-27a). Instead of choline, alcohols such as ethanolamine, serine, and the sugar derivative inositol are linked to the phosphate in other phosphoglycerides (Figure 5-28). The negative charge on the phosphate as well as the charged groups or hydroxyl groups on the alcohol esterified to it interact strongly with water. Both of the fatty acyl side chains in a phosphoglyceride may be saturated or unsaturated, or one chain may be saturated and the other unsaturated.

Sphingomyelin, a phospholipid that lacks a glycerol backbone, is found mainly in plasma membranes (Figure 5-27b). Instead of a glycerol backbone, it contains sphingosine, an amino alcohol with a long unsaturated hydrocarbon chain. In sphingomyelin, the terminal hydroxyl group of sphingosine is esterified to phosphocholine, so its hydrophilic head is similar to that of phosphatidylcholine.

Cholesterol and its derivatives constitute another important class of membrane lipids, the steroids. The basic structure of steroids is the four-ring hydrocarbon shown in Figure 5-29a. Cholesterol, the major steroidal constituent of animal tissues, has a hydroxyl substituent on one ring (Figure 5-29b). Although cholesterol is almost entirely hydrocarbon in composition, it is amphipathic because its hydroxyl group can interact with water. Cholesterol is especially abundant in the plasma membrane of mammalian cells but is absent from most prokaryotic cells. As much as 30 to 50 percent of the lipids in plant plasma membranes consists of cholesterol and certain steroids unique to plants.

Carbohydrates are found in many membranes, covalently bound either to proteins as constituents of glycoproteins or to lipids as constituents of glycolipids (see Figure 3-32). Bound carbohydrates increase the hydrophilic character of lipids and proteins and help to stabilize the conformations of many membrane proteins. The simplest glycolipid, glucosylcerebroside, contains a single glucose unit attached to a ceramide (Figure 5-27c).

Every Cellular Membrane Forms a Closed Compartment and Has a Cytosolic and an Exoplasmic Face

Phospholipids of the composition present in cells spontaneously form symmetric sheetlike phospholipid bilayers, which are two molecules thick. The hydrocarbon side chains in each leaflet form a hydrophobic core that is 3 – 4 nm thick in most biomembranes (Figure 5-30.) The various phospholipids differ in the charge carried by the polar head groups at neutral pH: some phosphoglycerides (e.g., phosphatidylcholine and phosphatidylethanolamine) have no net electric charge; others (e.g., phosphatidylglycerol and phosphatidylserine) have a net negative charge. Nonetheless, the polar head groups in all phospholipids can pack together into the characteristic bilayer structure. Sphingomyelins are similar in shape to phosphoglycerides and can form mixed bilayers with them.

Perhaps the most important lesson gleaned from the study of pure phospholipid bilayer membranes is that they spontaneously seal to form closed structures that separate two aqueous compartments (see Figure 2-20). Were a phospholipid bilayer to form a sheet with ends in which the hydrophobic interior were in contact with water, it would be unstable; thus a spherical structure with no ends is the most stable state of a phospholipid bilayer.

Similarly, all cellular membranes are closed structures, surrounding the cell itself or individual compartments. Cellular membranes thus have an internal face (the side oriented toward the interior of the compartment) and an external face (the side presented to the environment). Because most organelles are surrounded by a single bilayer membrane, it is also useful to speak of the cytosolic face and exoplasmic face of the membrane, the cytosol being the part of the cytoplasm outside of organelles (Figure 5-31). Thus the exoplasmic face of such organelles faces inward. Similarly, the exoplasmic face of the plasma membrane is directed away from the cytosol, in this case toward the extracellular space, and defines the outer limit of the cell. Some organelles, such as the nucleus, mitochondrion, and chloroplast, are surrounded by two membranes; in these cases, the exoplasmic surface faces the lumen, or space, between the two membranes.

Several Types of Evidence Point to the Universality of the Phospholipid Bilayer

A typical cell contains myriad types of membranes, each in turn bearing unique properties bestowed by its particular mix of lipids and proteins. When samples of plasma, nuclear, and mitochondrial membranes are prepared, using the cell-fractionation techniques we have already described, these preparations are often contaminated with the membranes of many other organelles. (The plasma membrane of human erythrocytes, however, can be isolated in near purity because these cells contain no internal membranes.) And all cellular membranes, regardless of their source, possess enormously varied protein-to-lipid ratios. The inner mitochondrial membrane, for example, is 76 percent protein; the myelin membrane, only 18 percent. The high phospholipid content of myelin allows it to electrically insulate the nerve cell from its environment.

Given the variable composition of cellular membranes, how certain are we that the phospholipid bilayer structure is common to all biomembranes? One piece of evidence is that many of the physical properties of pure phospholipid bilayers are similar to those of natural cellular membranes. Another is that either a single species of phospholipid, or a mixture of phospholipids with a composition approximating that found in natural membranes, spontaneously forms either planar bilayers or liposomes when dispersed in aqueous solutions (Figure 5-32).

Perhaps the best evidence for the bilayer structure comes from low-angle x-ray diffraction analysis of the multimembrane myelin sheath, which is elaborated by Schwann cells and covers and insulates many mammalian nerve cells (Figure 5-33a). The myelin sheath, which is a series of stacked membranes, is the major membrane component of such nerves and can be separated from other cellular membranes in a pure state, permitting direct physical and chemical analyses. X-ray diffraction analysis of these stacked plasma membranes has revealed a regular variation in density that is consistent with a bilayer organization of each membrane unit (Figure 5-33b). In this organization, protein is located mainly on either side of the membrane, which has hydrophilic external faces and a central region of almost pure low-density hydrocarbon. Although some polypeptide segments pass through the lipid bilayer, these make up less than 10 percent of the inner mass of the membrane and are not detected in this type of analysis.

Electron microscopy of thin membrane sections stained with osmium tetroxide, which binds strongly to the polar head groups of phospholipids, provides the most direct evidence for the universality of the bilayer structure. A cross section of all single membranes stained with osmium tetroxide looks like a railroad track: two thin dark lines (the stain – head group complexes) with a uniform light space of about 2 nm (the hydrophobic tails) between them (Figure 5-34). Although some osmium tetroxide may bind to double bonds in the hydrophobic fatty acyl chains, most of it binds to the polar head groups.

All Integral Proteins and Glycolipids Bind Asymmetrically to the Lipid Bilayer

The spaces inside and outside the closed compartments formed by cellular membranes usually have very different compositions. Such asymmetry is an essential aspect of the structure and function of biological membranes, and is reflected in the asymmetric structure of all integral membrane proteins. That is, each type of integral membrane protein has a single, specific orientation with respect to the cytosolic and exoplasmic faces of a cellular membrane, and all molecules of any particular integral membrane protein share this orientation (see Figure 3-33). This absolute asymmetry in protein orientation confers different properties on the two membrane faces. Proteins have never been observed to flip-flop across a membrane; such movement, involving a transient movement of hydrophilic amino acid and sugar residues through the hydrophobic interior of the membrane, would be energetically unfavorable. Accordingly, the asymmetry of a membrane protein, which is established during its biosynthesis and insertion into a membrane, is maintained throughout the protein’s lifetime.

Both glycoproteins and glycolipids are especially abundant in the plasma membrane of eukaryotic cells but are absent from the inner mitochondrial membrane, the chloroplast lamellae, and several other intracellular membranes. Almost invariably, attached carbohydrates are localized to the exoplasmic membrane face. Glycolipids are always found in the exoplasmic leaflet of membranes and are situated mainly, but not exclusively, on the surface membrane of cells. As with glycoproteins, their polar carbohydrate chains face outward toward the environment and away from the cell.

The Phospholipid Composition Differs in Two Membrane Leaflets

Most kinds of phospholipid, as well as cholesterol, are generally present in both membrane leaflets, although they are often more abundant in one or the other. For instance, in plasma membranes from human erythrocytes and certain canine kidney cells grown in culture, almost all the sphingomyelin and phosphatidylcholine, both of which have a positively charged head group (see Figure 5-27a, b), are found in the exoplasmic leaflet. In contrast, lipids with neutral or negative polar head groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol) are preferentially located in the cytosolic leaflet. Phosphorylated forms of phosphatidylinositol are cleaved as a result of cell stimulation by certain hormones, generating in the cytosol soluble forms of the “head groups” that affect many aspects of cellular metabolism (Chapter 20).

The relative abundance of a particular phospholipid in the two leaflets of a plasma membrane can be determined based on its susceptibility to hydrolysis by phospholipases, enzymes that cleave the phosphoester bonds that connect the phospholipid head groups (see Figure 3-37). Phospholipids in the cytosolic leaflet are resistant to hydrolysis by phospholipases added to the external medium, because the enzymes cannot penetrate to the cytosolic face of the plasma membrane. It is not clear how these differences in lipid composition of the two leaflets arise. One possibility is that certain lipids bind to specific protein domains that occur preferentially in one membrane leaflet.

Most Lipids and Integral Proteins Are Laterally Mobile in Biomembranes

In both pure phospholipid bilayers and natural membranes, thermal motion permits phospholipid and glycolipid molecules to rotate freely around their long axes and to diffuse laterally within the membrane leaflet. Because such movements are lateral or rotational, the fatty acyl chains remain in the hydrophobic interior of the membrane. In both natural and artificial membranes, a typical lipid molecule exchanges places with its neighbors in a leaflet about 10⁷ times per second and diffuses several micrometers per second at 37 °C. At this rate, a lipid could diffuse the length of a typical bacterial cell (≈1 μm) in only 1 second and the length of an animal cell in about 20 seconds.

In pure phospholipid bilayers, phospholipids do not migrate, or flip-flop, from one leaflet of the membrane to the other. In some natural membranes, however, they occasionally do so, catalyzed by certain membrane proteins called flippases (Chapter 15). Energetically, such movements are extremely unfavorable, because the polar head of a phospholipid must be transported through the hydrophobic interior of the membrane.

Various experiments have shown that many integral membrane proteins, like phospholipids, float quite freely within the plane of a natural membrane. In one such study, outlined in Figure 5-35, two different cells (e.g., mouse and human fibroblasts) are fused and the movement of their distinct surface proteins is then monitored at various times after incubation at 37 °C. Such experiments suggest that many integral proteins are free to diffuse in a sea of lipid in the two-dimensional space of the membrane. According to this concept, known as the fluid mosaic model, the membrane is viewed as a two-dimensional mosaic of laterally mobile phospholipid and protein molecules (see Figure 3-32). As discussed in Chapter 3, some integral membrane proteins consist of two or more noncovalently linked subunits; such multimeric membrane proteins float as a unit in the lipid.

The lateral movements of surface proteins and lipids can be quantified by a technique called fluorescence recovery after photobleaching (FRAP). With this method, described in Figure 5-36, the rate at which surface protein or lipid molecules move — the diffusion coefficient — can be determined, as well as the proportion of the molecules that are laterally mobile. FRAP studies with fluorescent-labeled phospholipids have shown that in fibroblast plasma membranes, all the phospholipids are freely mobile over distances of about 0.5 μm, but most cannot diffuse over much longer distances. These findings suggest that protein-rich regions of the plasma membrane, about 1 μm in diameter, separate lipid-rich regions containing the bulk of the membrane phospholipid. Phospholipids are free to diffuse within such a region 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 10⁻⁸ cm²/s and 10⁻⁷ 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.

Numerous experiments similar to those just discussed have shown that depending on the cell type, 30 – 90 percent of all integral proteins in the plasma membrane are freely mobile. Immobile proteins are permanently attached to the underlying cytoskeleton. The lateral diffusion rate of a mobile protein in an intact membrane is generally 10 – 30 times lower than that of the same protein embedded in synthetic liposomes. These findings suggest that the mobility of integral proteins in intact membranes is restricted by interactions with the rigid submembrane cytoskeleton. Clearly, such interactions would have to be broken and remade as mobile proteins diffuse laterally in the plasma membrane. Chapter 18 details the interactions of specific plasma membrane proteins with the underlying cytoskeleton, explaining how these affect cell shape and motility.

Fluidity of Membranes Depends on Temperature and Composition

One consequence of the packing of the fatty acyl chains within the center of a phospholipid bilayer is an abrupt change in its physical properties over a very narrow temperature range. For example, when a suspension of liposomes or a planar bilayer composed of a single type of phospholipid is heated, it passes from a highly ordered, gel-like state to a more mobile fluid state (Figure 5-37). During this phase transition, a relatively large amount of heat (thermal energy) is absorbed over a narrow temperature range; the midpoint of this range is the “melting temperature” of the bilayer.

In general, lipids with short or unsaturated fatty acyl chains undergo the phase transition at lower temperatures than do lipids with long or saturated chains. Compared with long chains, short chains have less surface area to form van der Waals interactions with one another. Since the gel state is stabilized by these interactions, short-chain lipids melt at lower temperatures than long-chain lipids. Likewise, the kinks in unsaturated fatty acyl chains (see Figure 2-18) result in their forming less stable van der Waals interactions with other lipids than do saturated chains. As a result, unsaturated lipids maintain a more random, fluid state at lower temperatures than lipids with saturated fatty acyl chains.

The hydrophobic interior of natural membranes generally has a low viscosity and a fluidlike, rather than gel-like, consistency. Maintenance of this bilayer fluidity appears to be essential for normal cell growth and reproduction. All cell membranes contain a mixture of different fatty acyl chains and are fluid at the temperature at which the cell is grown. Animal and bacterial cells adapt to a decrease in growth temperature by increasing the proportion of unsaturated to saturated fatty acids in the membrane, which tends to maintain a fluid bilayer at the reduced temperature.

Membrane cholesterol is another major determinant of bilayer fluidity. Cholesterol is too hydrophobic to form a sheet structure on its own, but it is intercalated (inserted) among phospholipids. Its polar hydroxyl group is in contact with the aqueous solution near the polar head groups of the phospholipids; the steroid ring interacts with and tends to immobilize their fatty acyl chains. The net effect of cholesterol on membrane fluidity varies, depending on the lipid composition. Cholesterol restricts the random movement of the polar heads of the fatty acyl chains, which are closest to the outer surfaces of the leaflets, but it separates and disperses their tails, causing the inner regions of the bilayer to become slightly more fluid. At the high concentrations found in eukaryotic plasma membranes, cholesterol tends to make the membrane less fluid at growth temperatures near 37 °C. Below the temperature that causes a phase transition, cholesterol keeps the membrane in a fluid state by preventing the hydrocarbon fatty acyl chains of the membrane lipids from binding to one another, thereby offsetting the drastic reduction in fluidity that would otherwise occur at low temperatures.

Membrane Leaflets Can Be Separated and Each Face Viewed Individually

When a frozen tissue specimen is fractured by a sharp blow, the fracture line frequently runs through the hydrophobic interior of cell membranes, separating the two phospholipid leaflets. Integral membrane proteins generally remain associated with one or the other leaflet of membranes subjected to this freeze-fracturing technique (Figure 5-38). The fractured specimen then is placed in a vacuum and the surface ice is removed by sublimation, a technique called deep etching or freeze etching. After metal shadowing with platinum and carbon, the organic material is removed by acid, leaving a carbon-metal replica of the membrane leaflet (see Figure 5-18).

Electron microscopy of membrane samples prepared by these techniques reveals numerous protuberances, most of which are membrane proteins (Figure 5-39). In deep-etching studies, the cytoplasmic face of a membrane is customarily called the P (protoplasmic) face and the exoplasmic face is the E face. It is not unusual for most or all protuberances to be on one of the two surfaces and their mirror images, in the form of pits or holes, to be on the other. This may occur because the integral proteins are bound more tightly to the lipids in one leaflet than to those in the other.

The Plasma Membrane Has Many Common Functions in All Cells

In all cells, the plasma membrane has several essential functions. These include transporting nutrients into and metabolic wastes out of the cell; preventing unwanted materials in the extracellular milieu from entering the cell; preventing loss of needed metabolites and maintaining the proper ionic composition, pH (≈7.2), and osmotic pressure of the cytosol. To carry out these functions, the plasma membrane contains specific transport proteins that permit the passage of certain small molecules but not others. Several of these proteins, discussed in detail in Chapter 15, use the energy released by ATP hydrolysis to pump ions and other molecules into or out of the cell against their concentration gradients. Small charged molecules such as ATP and amino acids can diffuse freely within the cytosol but are restricted in their ability to leave or enter it across the plasma membrane.

In addition to these universal functions, the plasma membrane has other crucial roles in multicellular organisms. Few of the cells in multicellular plants and animals exist as isolated entities; rather, groups of cells with related specializations combine to form tissues (Chapter 22). Specialized areas of the plasma membrane contain proteins and glycolipids that form specific contacts and junctions between cells to strengthen tissues and to allow the exchange of metabolites between cells. Other proteins in the plasma membrane act as anchoring points for many of the cytoskeletal fibers that permeate the cytosol, imparting shape and strength to cells. Surrounding most animal cells is a mixture of fibrous proteins and polysaccharides collectively called the extracellular matrix. This viscous, water-filled matrix provides a bedding on which most sheets of epithelial cells or small glands lie. Proteins in the plasma membrane anchor cells to many of the matrix components, adding to the strength and rigidity of many tissues (Figure 5-40). In addition, enzymes bound to the plasma membrane catalyze reactions that would occur with difficulty in an aqueous environment. The plasma membrane of many types of eukaryotic cells also contains receptor proteins that bind specific signaling molecules (e.g., hormones, growth factors, neurotransmitters), leading to various cellular responses. These membrane proteins, which are critical for cell development and functioning, are described in later chapters.

Unlike animal cells, plant cells are surrounded by a cell wall and lack the extracellular matrix found in animal tissues. As a plant cell matures, new layers of wall are laid down just outside the plasma membrane, which is intimately involved in the assembly of cell walls (Figure 5-41). The walls are built primarily of cellulose, a rodlike polysaccharide formed from β(1→14)-linked glucose monomers. The cellulose molecules aggregate, by hydrogen bonding, into bundles of fibers; other polysaccharides within the wall cross-link the cellulose fibers (Chapter 22). In woody plants, a complex water-insoluble polymer of phenol and other aromatic monomers, called lignin, imparts strength and rigidity to the cell walls. Other chemicals also are found in the walls of various plant cells; for example, waxes prevent plant tissues and proteins from drying out.

Like the entire cell, each organelle in eukaryotic cells is bounded by a membrane containing a unique set of proteins essential for its proper functioning. In the next section, we discuss the structure and function of the main organelles found in eukaryotic cells.

SUMMARY

• In a phospholipid bilayer, the long fatty acyl side chains in each leaflet are oriented toward one another, forming a hydrophobic core; the polar head groups line both surfaces (see Figure 5-30).

• The phospholipid bilayer forms the basic structure of all biomembranes, which also contain proteins, glycoproteins, cholesterol and other steroids, and glycolipids. The presence of specific sets of membrane proteins permits each type of membrane to carry out distinctive functions.

• All cellular membranes line closed compartments and have a cytosolic and an exoplasmic face (see Figure 5-31).

• The asymmetry of biological membranes is reflected in the specific orientation of each type of integral and peripheral membrane protein with respect to the cytosolic and exoplasmic faces. The presence of glycolipids exclusively in the exoplasmic leaflet also contributes to membrane asymmetry.

• Most integral proteins and lipids are laterally mobile in biomembranes. According to the fluid mosaic model, the membrane is viewed as a two-dimensional mosaic of phospholipid and protein molecules.

• As a phospholipid bilayer is heated, it undergoes a phase transition from a gel-like to a more fluid state over a short temperature range.

• Cholesterol is a major determinant of bilayer fluidity, although its effect depends on the composition of a membrane. Natural biomembranes generally have a fluid-like consistency, and cells adjust their phospholipid composition to maintain bilayer fluidity.

• In all cells, proteins in the plasma membrane selectively absorb nutrients, expel wastes, and maintain the proper intracellular ionic composition. Proteins in the plasma membrane anchor the membrane to intracellular cytoskeletal fibers and the extracellular matrix or cell wall. In multicellular organisms, plasma membrane proteins also act in the interactions and communication between cells, which are critical for proper functioning of multicellular tissues.

• In plants, the cell wall, which is built mainly of cellulose, is the major determinant of cell shape and imparts rigidity to cells.

• Animal cells, which lack a wall, are surrounded by an extracellular matrix consisting of collagen, glycoproteins, and other components that give strength and rigidity to tissues and organs.