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MOLECULAR CELL BIOLOGY SEVENTH EDITION

CHAPTER 10 Biomembrane Structure

Copyright © 2013 by W. H. Freeman and Company

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Figure 10-1 Molecular Biology of the Cell (© Garland Science 2008)

1. Function of membrane lipids Physical boundary

2. Function of membrane proteins 1) Channel or pump 2) Receptor 3) Structural link for cell architecture 4) Enzyme

Outer leaflet Inner leaflet

Figure 10.1 Fluid mosaic model of biomembranes.

Figure 10.3 The bilayer structure of biomembranes.

Lipid bilayer 1. Lipid bilayer determines the basic structure of biological membranes; while proteins are responsible for most membrane functions, serving as specific receptors, enzymes, transport proteins, etc (Figure 10-1 Molecular Biology of the Cell). 2. Phospholipids make up the majority of the non-protein components of biological membranes, and comprise the “fluid” aspect of the fluid-mosaic model (Figure 10-1). 3. The characteristic “railroad track” appearance of the membrane indicates the presence of two polar layers, consistent with the bilayer structure for phospholipid membranes. Schematic interpretation of the phospholipid bilayer in which polar groups face outward to shield the hydrophobic fatty acyl tails from water. The amphipathic nature of phospholipids, which governs their interactions, is critical 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 and sheetlike, two-molecule-thick phospholipid bilayers (Figure 10-3).

Figure 10.8 Three classes of membrane lipids.

Membrane lipids A typical biomembrane is assembled from phosphoglycerides, sphingolipids, and steroids. All three classes of lipids are amphipathic molecules having a polar (hydrophilic) head group and hydrophobic tail (Figure 10-8).

1. phosphoglicerides CH2OH CHOH + 3 CH3-(CH2)n-COOH CH2OH (fatty acid) ↓ ester bond CH2O-CO-(CH2)n-CH3 CHO-CO-(CH2)n-CH3 triacylglycerol CH2O-CO-(CH2)n-CH3 ↓ CH2O-CO-(CH2)n-CH3 CHO-CO-(CH2)n-CH3 phosphatidic acid CH2O-PO3

- ↓ alcohol CH2O-CO-(CH2)n-CH3 CHO-CO-(CH2)n-CH3 phospholipid CH2O-PO2-O-alcohol

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Phosphoglycerides 1. Phosphoglycerides, the most abundant class of lipids in most membranes, are derivatives of glycerol 3-phosphate. A typical phosphoglyceride molecule consists of a hydrophobic tail composed of two fatty acyl chains esterified to the two hydroxyl groups in glycerol phosphate and a polar head group attached to the phosphate group. At neutral pH, some phosphoglycerides (e.g., phosphatidylcholine and phosphatidylethanolamine) carry no net electric charge, whereas others (e.g., phosphatidylinositol and phosphatidylserine) carry a single net negative charge. Nonetheless, the polar head groups in all phospholipids can pack together into the characteristic bilayer structure. 2. A phosphogyceride is classified according to the nature of its head group. In phosphatidylcholines, the most abundant phospholipids in the plasma membrane, the head group consists of choline, a positively charged alcohol, esterified to the negatively charged phosphate. In other phosphoglycerides, an OH-containing molecule such as ethanolamine, serine, and the sugar derivative inositol is linked to the phosphate group. The negatively charged phosphate group and the positively charged groups or the hydroxyl groups on the head group interact strongly with water (Table 2-5).

1) C #: 12-26 2) C1: saturated f.a. (동물성) C2: unsaturated f.a (식물성) 3) double bond #:<6 chain 중간, cis 形

Fatty acid

UN Figure 2.12

UN Figure 2.12

Figure 2.21 The effect of a double bond on the shape of fatty acids.

Fatty acid 1. Fatty acids consist of a hydrocarbon chain attached to a carboxyl group (-COOH). They differ in length, although the predominant fatty acids in cells have an even number of carbon atoms, usually 14, 16, 18, or 20. The major fatty acids in phospholipids are listed in Table 2-4. Fatty acids often are designated by the abbreviation Cx:y, where x is the number of carbons in the chain and y is the number of double bonds. Fatty acids containing 12 or more carbon atoms are nearly insoluble in aqueous solutions because of their long hydrophobic hydrocarbon chains. 2. Fatty acids with no carbon-carbon double bonds are said to be saturated; those with at least one double bond are unsaturated. Unsaturated fatty acids with more than one carbon-carbon double bond are referred to as polyunsaturated. Two “essential” polyunsaturated fatty acids, linoleic acid (C18:2) and linolenic acid (C18:3), cannot be synthesized by mammals and must be supplied in their diet. Mammals can synthesize other common fatty acids. Two stereoisomeric configurations, cis and trans, are possible around each carbon-carbon double bond: A cis double bond introduces a rigid kink in the otherwise flexible straight chain of a fatty acid. In general, the fatty acids in biological systems contain only cis double bonds (UN Figure 2-12). 3. In chemical structure, arachidonic acid is a carboxylic acid with a 20-carbon chain and four cis-double bonds; the first double bond is located at the sixth carbon from the omega end. In addition to being involved in cellular signaling as a lipid second messenger involved in the regulation of signaling enzymes, such as PLC-γ, PLC-δ, and PKC-α, -β, and -γ isoforms, arachidonic acid is a key inflammatory intermediate and can also act as a vasodilator. 4. A cis double bond introduces a rigid kink in the otherwise flexible straight chain of a fatty acid. In general, the fatty acids in biological systems contain only cis double bonds (Figure 2-21).

UN Figure 2.9

Triacylglycerol Fatty acids can be covalently attached to another molecule by a type of dehydration reaction called esterification, in which the OH from the carboxyl group of the fatty acid and a H from a hydroxyl group on the other molecule are lost. In the combined molecule formed by this reaction, the portion derived from the fatty acid is called an acyl group, or fatty acyl group. This is illustrated by triacylglycerols, which contain three acyl groups esterfied to glycerol (UN Figure 2-9).

Figure 10.12 Specificity of phospholipases.

Phospholipases The relative abundance of a particular phospholipid in the two leaflets of a plasma membrane can be determined on the basis of its susceptibility to hydrolysis by phospholipases, enzymes that cleave various bonds in the hydrophilic ends of phospholipids (Figure 10-12).

2. Sphingolipid serine ↓ CHOH-CH=CH-(CH2)12-CH3

CH-NH3+ sphingosine

CH2OH ↓ 1 fatty acid CHOH-CH=CH-(CH2)12-CH3

CH-NH2-CO-(CH2)n-CH3 ceramide CH2OH ↓ phosphocholine CHOH-CH=CH-(CH2)12-CH3 CH-NH2-CO-(CH2)n-CH3 sphingomyelin CH2O-phosphocholine

COO- CH-NH3

+ CH2OH

Sphingomyelinase: ceramide와 phosphocholine 생성

Sphingolipid 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 to the sphingosine amino group. In sphingomyelin, the most abundant sphingolipid, phosphocholine is attached to the terminal hydroxyl group of sphingosine. Thus sphingomyelin is a phospholipid, and its overall structure is quite similar to that of phosphatidylcholine. Acid Sphingomyelinase is one of the enzymes that make up the Sphingomyelinase (SMase) family, responsible for catalyzing the breakdown of sphingomyelin to ceramide and phosphorylcholine. These results underline the importance of diacylglycerol in the regulation of programmed cell death and strongly argue for a balance between apoptotic (ceramide) and survival (diacylglycerol) signal transducers.


3. Cholesterol - 막 구성성분 - 담즙산 - steroid hormone


Cholesterol Although phospholipids are critical for the formation of the classic bilayer structure of membranes, eukaryotic cell membranes require other components, including sterols. Here, we focus on cholesterol, the principal sterol in animal cells and the most abundant single lipid in the mammalian plasma membrane (almost equimolar with all phospholipids). Between 50 and 90 percent of the cholesterol in most mammalian cells is present in the plasma membrane and related endocytic vesicle membranes. Cholesterol is also critical for intercellular signaling and has other functions to be described shortly. Cholesterol and its derivatives constitute the third important class of membrane lipids, the steroids. The basic structure of steroids is a four-ring hydrocarbon. Cholesterol, the major steroidal constituent of animal tissues, has a hydroxyl substituent on one ring. 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 membranes of mammalian cells but is absent from most prokaryotic cells. Excess cholesterol is converted by liver cells into bile acids (e.g., deoxycholic acid), which are secreted into the bile. Specialized endocrine cells synthesize steroid hormones (e.g., testosterone) from cholesterol, and photochemical and enzymatic reactions in the skin and kidneys produce vitamin D (Figure 10-4, 10-5 Molecular Biology of the Cell).


Membrane proteins 1) integral (intrinsic) protein: 막을 관통하거나 lipidation 되어 있음 분리: detergent (SDS, Triton X-100) 2) peripheral (extrinsic) protein: 인지질의 head나 integral protein에 비공유결합됨 분리: pH 변화, high/low salt

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Membrane proteins 1. Integral proteins containing membrane-spanning α-helical domains are embedded in membranes by hydrophobic interactions with specific lipids and probably also by ionic interactions with the polar head groups of the phospholipids. 2. Peripheral proteins associate with the membrane primarily by specific noncovalent interactions with integral proteins or membrane lipids (Figure 10-19 Molecular Biology of the Cell).

Glycophorin A: 20-30(23) hydrophobic AA sequence, alpha-helix

Figure 10.14 Structure of glycophorin A, a typical single-pass transmembrane protein.

Figure 10.14 Structure of glycophorin A, a typical single-pass transmembrane protein.

Glycophorin A Glycophorin A, the major protein in the erythrocyte plasma membrane, is a representative single-pass transmembrane protein, which contains only one membrane-spanning α helix. Typically, a membrane-embedded helix is composed of 20–25 hydrophobic (uncharged) amino acids. 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 domain is heavily glycosylated, with the carbohydrate side chains (green diamonds) attached to specific serine, threonine, and asparagine residues (Figure 10-14).


Bacteriorhodopsin of Halobacterium light-activated H+ pump for ATP production

Bacteriorhodopsin of Halobacterium Structural model of bacteriorhodopsin, a multipass transmembrane protein that functions as a photoreceptor in certain bacteria. The seven hydrophobic α helices in bacteriorhodopsin traverse the lipid bilayer. Absorption of light by the retinal group covalently attached to bacteriorhodopsin 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 (Figure 10-32 Molecular Biology of the Cell, Figure 10-15).

Figure 10.19 Anchoring of plasma-membrane proteins to the bilayer by covalently linked hydrocarbon groups.

Anchoring of plasma-membrane proteins In eukaryotic cells, several types of covalently attached lipids anchor some proteins to one or the other leaflet of the plasma membrane and certain other cellular membranes. In these lipid-anchored proteins, the lipid hydrocarbon chains are embedded in the bilayer, but the protein itself does not enter the bilayer (Figure 10-19). 1. A group of cytosolic proteins are anchored to the cytosolic face of a membrane by a fatty acyl group

(e.g., myristate or palmitate) attached to the N-terminal glycine residue (a). 2. A second group of cytosolic proteins are anchored to membranes by an unsaturated fatty acyl

group attached to a cysteine residue at or near the C-terminus (b). 3. Some cell-surface proteins and heavily glycosylated proteoglycans of the extracellular matrix are

bound to the exoplasmic face of the plasma membrane by a third type of anchor group, glycosylphosphatidylinositol (GPI). The exact structures of GPI anchors vary greatly in different cell types, but they always contain phosphatidylinositol (PI), whose two fatty acyl chains extend into the lipid bilayer; phosphoethanolamine, which covalently links the anchor to the C-terminus of a protein; and several sugar residues (c).

1) lateral diffusion/rotation: 107 molecules/sec in pure phospholipid bilayer and cell membrane

2) flip-flop: <1/1 month in pure phospholipid

Mobility of membrane lipid

Figure 10-11b Molecular Biology of the Cell (© Garland Science 2008)

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Mobility of membrane lipid 1. 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 remain in the hydrophobic interior of the bilayer. In both natural and ar-tificial 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. 2. In pure bilayers, phospholipids do not spontaneously migrate, or flip-flop, from one leaflet to the other. Energetically, such flip-flopping is extremely unfavorable because it entails movement of the polar phospholipid head group through the hydrophobic interior of the membrane (Figure 10-11b Molecular Biology of the Cell).

Figure 10.9 Gel and fluid forms of the phospholipid bilayer.

Factors affecting membrane lipid fluidity 1) temperature↑ → fluidity ↑ gel to liquid-crystalline phase transition 2) acyl chain C # ↑ → fluidity ↓ 3) double bond # ↑ → fluidity ↑ cis 형>trans 형, position 중앙 4) cholesterol ↑ → fluidity ↓ ( at 37℃) 온도가 낮은 상태에서는 fluidity↑

Fluidity of the phospholipid bilayer The ability of lipids to diffuse laterally in a bilayer indicates that it can act as a fluid. The degree of bilayer fluidity depends on the lipid composition, structure of the phospholipid hydrophobic tails, and temperature. As already noted, van der Waals interactions and the hydrophobic effect cause the nonpolar tails of phospholipids to aggregate. Long, saturated fatty acyl chains have the greatest tendency to aggregate, packing tightly together into a gel-like state. Phospholipids with short fatty acyl chains, which have less surface area for interaction, form more fluid bilayers. Likewise, the kinks in unsaturated fatty acyl chains result in their forming less stable van der Waals interactions with other lipids than do saturated chains and hence more fluid bilayers. When a highly ordered, gel-like bilayer is heated, the increased molecular motions of the fatty acyl tails cause it to undergo a transition to a more fluid, disordered state (Figure 10-9).


Flip-flop of lipid in cell membranes

Figure 10.27 Proposed mechanisms of transport of cholesterol and phospholipids between membranes.

Flip-flop of lipid in cell membranes 1. Even though phospholipids are initially incorporated into the cytosolic leaflet of the ER membrane, various phospholipids are asymmetrically distributed in the two leaflets of the ER membrane and of other cellular membranes. However, phospholipids spontaneously flip-flop from one leaflet to the other only very slowly, although they can rapidly diffuse laterally in the plane of the membrane (Figure 12-58 Molecular Biology of the Cell). ① For the ER membrane to expand (growth of both leaflets) and have asymmetrically distributed

phospholipids, its phospholipid components must be able to rapidly and selectively flip-flop from one membrane leaflet to the other.

② The usual asymmetric distribution of phospholipids in membrane leaflets is broken down as cells (e.g., red blood cells) become senescent or undergo apoptosis. For instance, phosphatidylserine and phosphatidylethanolamine are preferentially located in the cytosolic leaflet of cellular membranes. Increased exposure of these anionic phospholipids on the exoplasmic face of the plasma membrane appears to serve as a signal for scavenger cells to remove and destroy old or dying cells. Although the mechanisms employed to generate and maintain membrane phospholipid asymmetry are not well understood, it is clear that flippases play a key role. These integral membrane proteins facilitate the movement of phospholipid molecules from one leaflet to the other.

2. Three mechanisms have been proposed for the transport of cholesterol and phospholipids from their sites of synthesis to other membranes independently of the Golgi-mediated secretory pathway (Figure 10-27) ① First, some Golgi-independent transport is most likely through membrane-limited vesicles or other

protein–lipid complexes. ② The second mechanism entails direct protein-mediated contact of ER or ER-derived membranes

with membranes of other organelles. ③ In the third mechanism, small lipid-transfer proteins facilitate the exchange of phospholipids or

cholesterol between different membranes.


막단백질의 유동성 실험 I : Frye와 Edidin의 실험 5

막단백질의 유동성 실험 I : Frye와 Edidin의 실험 1. Like membrane lipids, membrane proteins do not tumble (flip-flop) across the lipid bilayer, but they do rotate about an axis perpendicular to the plane of the bilayer(rotational diffusion). In addition, many membrane proteins are able to move laterally within the membrane (lateral diffusion). 2. The first direct evidence that some plasma membrane proteins are mobile in the plane of the membrane was provided by an experiment in which mouse cells were artificially fused with human cells to produce hybrid cells (heterocaryons). Two differently labeled antibodies were used to distinguish selected mouse and human plasma membrane proteins. Although at first the mouse and human proteins were confined to their own halves of the newly formed heterocaryon, the two sets of proteins diffused and mixed over the entire cell surface within half an hour or so. This same lateral fluidity was first demonstrated conclusively on the cell surface by Frye and Edidin in 1970. They fused two cells labeled with different membrane-bound fluorescent tags and watched as the two dye populations mixed. The results of this experiment were key in the development of the "fluid mosaic" model of the cell membrane by Singer and Nicolson in 1972. According to this model, biological membranes are composed largely of bare lipid bilayer with proteins penetrating either half way or all the way through the membrane. These proteins are visualized as freely floating within a completely liquid bilayer (Figure 10-35 Molecular Biology of the Cell).

Figure 10-36a Molecular Biology of the Cell (© Garland Science 2008)

막단백질의 유동성 실험 II : Fluorescence recovery after photobleaching

Experimental Figure 10.10 Fluorescence recovery after photobleaching (FRAP) experiments can quantify the lateral movement of proteins and lipids within the plasma membrane.

막단백질의 유동성 실험 II : Fluorescence recovery after photobleaching The lateral movements of specific plasma-membrane proteins and lipids can be quantified by a technique called fluorescence recovery after photobleaching (FRAP). With this method, described in Figure 10-36a Molecular Biology of the Cell and Figure 10-10, the rate at which membrane lipid or protein molecules move—the diffusion coefficient—can be determined, as well as the proportion of the molecules that are laterally mobile (Figure 10-36a Molecular Biology of the Cell, Experimental Figure 10-10). Step 1 : Cells are first labeled with a fluorescent reagent that binds uniformly to a specific membrane lipid or protein. Step 2: 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 3 : In time, the fluorescence 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 proportional to the fraction of labeled molecules that are mobile in the membrane.

Figure 10-36b Molecular Biology of the Cell (© Garland Science 2008)

막단백질의 유동성 실험 III: Fluorescence loss in photobleaching

막단백질의 유동성 실험 III: Fluorescence loss in photobleaching Lateral diffusion can also be measured by a complementary strategy know as fluorescence loss in photo-bleaching (FLIP). In this technique, a small area is continuously bleached and the fluorescent proteins are bleached as they diffuse into it. Eventually, the number of fluorescent proteins will decrease and will result in all bleached proteins. From both FRAP and FLIP, we can calculate the diffusion coefficient from the bleached proteins (Figure 10-36b Molecular Biology of the Cell).


Restricting the lateral mobility of membrane proteins 6

Tight junction in intestinal epithelial cell

Restricting the lateral mobility of membrane proteins 1. There are restrictions to the lateral mobility of the lipid and protein components in the fluid membrane imposed by the formation of subdomains within the lipid bilayer. These subdomains arise by several processes e.g. binding of membrane components to the extracellular matrix, nanometric membrane regions with a particular biochemical composition that promote the formation of lipid rafts and protein complexes mediated by protein-protein interactions. Furthermore, protein-cytoskeleton associations mediate the formation of “cytoskeletal fences”, corrals wherein lipid and membrane proteins can diffuse freely, but that they can seldom leave. Restriction on lateral diffusion rates of membrane components is very important because it allows the functional specialization of particular regions within the cell membranes (Figure 10-39 Molecular Biology of the Cell). ① The proteins can self-assemble into large aggregates (as seen for bacteriorhodopsin in the

purple membrane of Halobacterium); ② they can be tethered by interactions with assemblies of macromolecules (B) outside or (C) inside

the cell; ③ or they can interact with proteins on the surface of another cell (D). 2. Tight junctions, lying just under the microvilli, prevent the diffusion of many substances through the extracellular spaces between the cells. They also maintain the polarity of epithelial cells by preventing the diffusion of membrane proteins and glycolipids (lipids with covalently attached sugars) between the apical and the basolateral regions of the plasma membrane, ensuring that these regions contain different membrane components.

Figure 10.23 Solubilization of integral membrane proteins by nonionic detergents.

Detergent A class of lipid molecule having amphipathic structure but with high ratio of hydrophilic to hydrophobic properties

Figure 10.22 Structures of four common detergents.

Detergent Detergents are amphipathic molecules that disrupt membranes by intercalating into phospholipid bilayers and solubilizing lipids and proteins. 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 charged group (Figure 10-22). At very low concentrations, detergents dissolve in pure water as isolated molecules. As the concentration increases, the molecules begin to form micelles—small, spherical aggregates in which hydrophilic parts of the molecules face outward and the hydrophobic parts cluster in the center. The critical micelle concentration (CMC) at which micelles form is characteristic of each detergent and is a function of the structures of its hydrophobic and hydrophilic parts (Figure 10-23).

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Artificial membranes 1) Black lipid membrane 2) Liposome

Experimental Figure 10.4 Formation and study of pure phospholipid bilayers

Artificial membrane: study on function of membrane proteins


Artificial membranes 1. Lipid bilayers can be created artificially in the lab to allow researchers to perform experiments that cannot be done with natural bilayers. These synthetic systems are called model lipid bilayers. There are many different types of model bilayers, each having experimental advantages and disadvantages. The first system developed was the black lipid membrane or “painted” bilayer, which allows simple electrical characterization of bilayers but is short-lived and can be difficult to work with. 2. Although transport proteins can be isolated from membranes and purified, the functional properties of these proteins can be studied only when they are associated with a membrane. To facilitate such studies, researchers use two approaches for enriching a transport protein of interest so that it predominates in the membrane. In one common approach, a specific transport protein is extracted and purified; the purified protein then is reincorporated into pure phospholipid bilayer membranes, such as liposomes (Experimental Figure 10-4, Figure 10-31 Molecular Biology of the Cell).

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