Suggested Reading: Stryer Chapter 11.
A barrier that separates cellular contents from the environment is an absolute condition for life. This separation is provided by the plasma membrane also called the cell membrane. Plasma membranes are selective permeability barriers that mediate the flow of molecules into and out of the cell, and in bacteria, participate in the generation of metabolic energy. They also contain molecules at their surfaces that are involved in cellular recognition and communication. Eukaryotic cells also contain many internal membrane systems that define the boundaries of cell organelles. Each internal membrane is specialized to assist the function of the organelle it surrounds.
I. The Chemistry of Lipids
Membranes are all comprised of lipids and proteins. Although lipids display considerable structural diversity, they share one important property: they are all amphipathic. One end of the lipid molecule (the head) is polar, and therefore hydrophilic; the other end, the tail, is non-polar, and thus hydrophobic. Lipids are only sparingly soluble in water. Three classes of lipids are found in the membranes of animal cells: (a) phospholipids [phosphate-containing lipids] (b) glycolipids [sugar-containing lipids] and (c) cholesterol.
Phospholipids and glycolipids have very similar structures, consisting of a fatty acid tail, a three-carbon backbone, and a polar head group. The fatty acid chains generally contain an even number of carbon atoms, usually ranging between 14 and 24 carbons in length. The most common fatty acids have 16 or 18 carbon-long, fatty acid chains, and 0-3 cis double bonds. The length of the fatty acid chains and the degree of unsaturation have important consequences with regard to the physical properties of membranes. Palmitate (C16, saturated), stearate (C18, saturated) and oleate (C18, one double bond) are frequently occurring fatty acids.
An unsaturated fatty acid with a cis double bond, such as oleate, has a pronounced bend in its hydrocarbon chain which prevents the formation of closely packed, well-ordered crystals. As a result, there are fewer van der Waals interactions among hydrocarbon chains in the crystal, which lowers the melting point of the fatty acid. Thus, the melting point of an unsaturated fatty acid is much lower than that of a saturated fatty acid (compare stearate with oleate). For the same reasons, longer fatty acids have higher melting temperatures than shorter ones (compare stearate with palmitate).
Glycerol and sphingosine are the two types of three-carbon backbone that occur in phospholipids and glycolipids. All phospholipids except sphingomyelin derive from glycerol and are called phosphoglycerides. In phosphoglycerides, two fatty acids are attached to C1 and C2 of glycerol by ester linkages to form diacylglycerol. Occasionally, glycerol is attached to a hydrocarbon chain through an ether linkage at C1, forming a special class of phosphoglycerides called plasmalogens.
The headgroup of phosphoglycerides results from phosphorylation at C3 of diacylglycerol to form phosphatidic acid. Phosphatidic acid is subsequently attached to several different polar alcohols by a phosphoester linkage to generate different phosphoglycerides. Three important phosphoglycerides are phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI). In the phosphoglyceride cardiolipin, glycerol appears in the head group and links two molecules of phosphatidic acid into a lipid dimer.
All glycolipids derive from sphingosine. Sphingosine differs from glycerol in two ways: (a) it contains a C15 hydrocarbon chain attached by a carbon-carbon bond at C1 (b) it has an amine rather than an alcohol at C2. Fatty acids are attached at C2 of sphingosine by amide linkages to form ceramides.
Glycolipids are formed by attachment of sugar head groups to C3 of ceramides through an O-glycosidic linkage. Cerebrosides have single sugar head groups (either glucose or galactose). Gangliosides have more complex oligosaccharide head groups that can include the negatively charged sugar sialic acid. Gangliosides containing sulfate-modified sugars are called sulfatides.
Sphingomyelin is the only phospholipid derived from sphingosine. It contains a phosphocholine group at C1 of ceramide attached by a phosphoester linkage.
Cholesterol is a component of eukaryotic (but not prokaryotic) membranes. It is mostly located in the plasma membrane. Cholesterol is synthesized from isoprene subunits (isopentyl pyrophosphate) and will be discussed in the lectures on lipid biosynthesis. It has a single alcohol head group and a steroid tail. Cholesterol adopts a rigid, plate-like structure that is shorter than the hydrocarbon tails of phospholipids and glycolipids.
II. Physical Properties of Lipid Structures.
Structures. The amphipathic nature of lipid molecules determines their structure: the hydrophobic regions group together to avoid water (hydrophobic effect) and form favorable van der Waals interactions; the hydrophilic groups interact strongly with water through ionic and hydrogen bonds. Three possible structures can accommodate both of these requirements: a monolayer at an air-water interface, a micelle, and a bilayer sheet.
Membrane assembly is spontaneous, and is driven by the physical interactions described above. Lipid bilayers fold on themselves so that no edges are exposed to water--this is how intracellular compartments are formed. They are also self-sealing, because a hole is energetically unfavorable. Biological membranes are typically 75 Å wide; the hydrocarbon core alone is 30 Å wide.The structure formed by a particular lipid depends on the shape of its van der Waals envelope. For wedge-shaped lipids, the headgroup requires a larger packing volume than the hydrocarbon tail; for cone-shaped lipids the hydrocarbon tail requires a larger packing volume than the headgroup; for cylinder-shaped lipids, the packing volume of the headgroup and hydrocarbon tail are similar. For example, the two fatty acid side chains of phospholipids are too bulky to form a stable micelle, thus a bilayer structure is favored. Alternatively, the salts of fatty acids (such as sodium palmitate, a component of soap) contain only one fatty acid chain, and thus readily form a micelle.
Permeability. Membrane bilayers are selective permeability barriers. The ability of a molecule to cross a membrane correlates well with its solubility in apolar solvents vs. water. As a rule of thumb, small hydrophobic molecules (e.g. indole) and small uncharged polar molecules (e.g. H2O) diffuse out of a liposome of 100 mM radius with a half-time of less than one minute, while large polar molecules (e.g. glucose) and ions (e.g. Cl-) diffuse out with half-times of many hours. The permeability of a molecule is described quantitatively by its permeability coefficient P. The outward flux of a small molecule across a bilayer, F, and the first order rate constant for its passive outward diffusion from a liposome, kperm, can be calculated from the permeability coefficient:
where Cinside and Coutside are the concentrations of the molecule inside and outside the bilayer and R is the radius of the liposome. Almost all biochemical metabolites cross the plasma membrane at negligible rates. One exception is indole, the side chain of tryptophan, which has a large permeability coefficient and can therefore escape from the cell. Nature has solved this problem by substrate channeling in a multi-enzyme complex. Indole synthesized by the a subunit of tryptophan synthase diffuses along a 25 Å protein channel to the b subunit, where it is attached to the polar amino-acid backbone. Indole is never allowed to diffuse freely in the cytosol.
Dynamics. Lipids move rapidly in the plane of the membrane bilayer by lateral diffusion. In this respect, a membrane behaves as a two-dimensional fluid. Lipid mobility is expressed in terms of a diffusion constant, Dlipid; the average distance s traversed in two dimensions by a lipid in time t depends on Dlipid according to:
A typical value of Dlipid is 1 mm2·s-1. Thus, a lipid molecule travels ~2 mm in a membrane bilayer in one second. At a much slower rate, lipids can diffuse between the leaflets of the lipid bilayer by a process called transverse diffusion or flip-flop. The rate at which a lipid undergoes transverse diffusion depends on the polarity of its head group, which must pass through the hydrocarbon core of the bilayer during the flip-flop process. Half-lives for transverse diffusion of lipids fall between hours to several months. For example, erythrocytes (red blood cells) exist in the peripheral circulation for ~120 days without carrying out membrane synthesis or repair. Nevertheless, no measurable loss of asymmetry in the lipid composition of the inner and outer leaflets of the plasma membrane (see below) occurs during this time. Biological membranes contain proteins called flippases which catalyze the flip-flop of specific lipids across bilayers. These proteins will be discussed further in the lectures on membrane biosynthesis.
Phase Transition. An artificial lipid bilayer made from a single type of phospholipid changes from a liquid state to a rigid crystalline state (or gel) at a sharp and characteristic freezing temperature (TF). This change of state is called a phase-transition, and the temperature at which it occurs is lower (that is, the membrane becomes more difficult to freeze) if the hydrocarbon chains are short or if they contain double bonds. A shorter chain length reduces the tendency of hydrocarbon tails to interact with each other, and cis-double bonds produce kinks in the chains that make it more difficult for them to pack together.
Cholesterol enhances the mechanical stability of the bilayer, and also is important in maintaining the fluidity of the bilayer. The plate-like steroid ring structure of cholesterol interacts with--and partly immobilizes--those regions of the hydrocarbon chains nearest to the polar head group, leaving the rest of the chain flexible. Cholesterol prevents hydrocarbon chains from coming together and crystallizing. Thus, cholesterol inhibits temperature-induced phase transitions and prevents the drastic decrease in membrane fluidity that would otherwise occur at low temperatures.
Gaseous anesthetics (diethyl ether, halothane and cyclopropane) and ethanol are thought to interfere with transmission of nerve signals by altering the membrane fluidity of cells in the central nervous system. Membrane fluidity can also be effected by diet.
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Membrane Composition |
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Longer Hydrocarbon Chains |
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Higher [Ca2+] |
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More cis Double Bonds |
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EtOH, Anesthetics |
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Increase [Cholesterol] |
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III. Membrane Proteins.
Biological membranes consist of 25-75% protein by weight. Each membrane in a cell contains a specialized set of proteins which assist the function of the compartment it surrounds. Membrane proteins transduce signals (e.g. insulin receptor), generate energy (e.g. F1F0 ATPase), actively and passively transport molecules (e.g. proton pumps) and provide mechanical support to the bilayer (e.g. glycophorin A).
Membrane proteins fall into three categories, transmembrane, lipoprotein, and peripheral, depending on how they associate with the lipid bilayer. The polypeptide chains of transmembrane proteins (TM-proteins) cross the lipid bilayer at least once. Lipoproteins are covalently linked to hydrocarbon chains in the bilayer. TM-proteins and lipoproteins are termed integral membrane proteins because they can only be released by disrupting the bilayer with detergents. Peripheral membrane proteins associate with the bilayer through protein-protein interactions and can be released by addition of mild denaturants, addition of salt, or variation of pH.
TM Proteins. The polypeptide chains of TM proteins must cross the 30 Å hydrocarbon core of a lipid bilayer without stranding any polar sidechains or backbone peptide groups. Nature utilizes two protein structural motifs to solve this difficult problem: the transmembrane helix and the b-barrel.
TM helices are formed by stretches of twenty contiguous hydrophobic amino acids that fold into a 30 Å a-helix. The hydrophobic sidechains displayed on the surface of TM helices are soluble in the hydrocarbon core of the bilayer. The polar backbone peptide groups are solvated by intra-molecular hydrogen bonds. TM helices are identified in novel protein sequences from the human genome sequencing project by looking for runs of nonpolar amino acids.
b-Barrels, exemplified in the structure of the porin protein, consist of large b-sheets that wrap around to close on themselves. As in TM helices, the polar backbone peptide groups are solvated by intramolecular hydrogen bonds. Hydrophobic sidechains face the hydrocarbon core of the bilayer.
Lipoproteins. Lipoproteins are linked to the plasma membrane by a number of different post-translational covalent modifications:
Inner Leaflet:
Myristic Acid (C14) --- Amide linkage to N-terminal glycine.
Palmitic Acid (C16) --- Ester/thiolester linkage to sidechains of Ser/Thr/Cys
Prenyl (isoprene based) --- Thioether linkage to CaaX-COO- (a=aliphatic)
Farnesyl (C15)
Geranyl (C20)
aaX is proteolytically removed and C esterified to methyl ester
Outer leaflet:
Glycosyl Phosphatidyl Inositol (GPI) --- Amide linkage to C-terminus
Protein-PE-Man3(Gal4)GlcNAc1-PI
Glycosylation of Membrane Proteins. Many proteins on the outer surface of the plasma membrane are extensively modified with sugar groups. Sugars are attached by N-glycosidic linkages to asparagine side chains and by O-glycosidic linkages to serine and threonine side chains. The proximal sugar is usually N-acetyl glucosamine (GlcNAc) or N-acetyl galactosamine (GalNAc). Proteins with oligosaccharide modifications are termed glycoproteins, while those with more extensive polysaccharide moieties are termed proteoglycans.
Dynamics of Membrane Proteins. The rate of lateral diffusion of membrane proteins varies greatly. The photoreceptor rhodopsin moves nearly as rapidly as lipids (D= 0.4 mM2/s) while the glycoprotein fibronectin, which interacts with the extracellular matrix, is virtually immobile (D< 10-4 mM2/s). Transverse diffusion of membrane proteins never occurs because polar amino acids in hydrated portions of the polypeptide chain prevent it.
Detergents. Detergents are amphipihilic molecules that have a propensity to form micelles. Chemically, detergents look like lipids with a single hydrocarbon tail, and have a wedge-shaped van der Waals envelope. The critical micelle concentration (CMC) of a detergent is the concentration of detergent monomers at which micelles begin to form. Detergents are used to disrupt biological membranes so that membrane proteins can be isolated in protein-containing micelles. Ideal detergents dissociate proteins from the other membrane components without unfolding them. The reconstitution of micelle-solubilized proteins into synthetic liposomes is a powerful approach to the study of membrane processes.
IV. Structures that Ramify the Plasma Membrane.
Plasma membranes are mechanically supported by the cell cortex on their cytosolic surface, and are lubricated by the glycocalyx on their exterior surface. These structures have been extensively studied in erythrocytes (red blood cells). Erythrocytes are subjected to mechanical stress as they are pumped through small passages in the peripheral circulation. The sole function of glycophorin, an abundant membrane protein in erythrocytes, is to participate in the glycocalyx and cortex structures.
Cells are Sugar-Coated. Sugars from heavily glycosylated membrane proteins and from glycolipids in the plasma membrane form the glycocalyx, a thick carbohydrate layer that covers the cell surface. The glycocalyx serves two important purposes. First, its oligosaccharide and polysaccharide constituents absorb water to form a slimy surface. This surface protects the membrane from mechanical and chemical damage and prevents the cell from sticking nonspecifically to other tissues (the walls of a blood vessel, for example!). Second, the glycocalyx functions as the cell’s uniform. In sperm-egg fertilization, homing of lymphocytes to parental tissues, and adhesion of neutrophils to sites of endothelial injury in the inflammatory response, cell-cell adhesion occurs by recognition of specific carbohydrate moieties in the glycocalyx. The proteins that recognize carbohydrate sequences are called lectins. The plant lectins concanavalin A (which recognizes terminal non-reducing a-mannose residues) and wheat germ agglutinin (which recognizes GlcNAc3) are frequently used as tools in the biochemistry laboratory. In humans, L (lymph), E (endothelium) and and P (platelet) selectins mediate carbohydrate recognition during clotting, inflammatory and immune responses.
The Membrane Skeleton. Plasma membranes are supported from beneath by the cell cortex, a protein mesh under the bilayer. In erythrocytes, the cortex consists primarily of spectrin, a filamentous protein composed of three-stranded coiled-coil domains. Spectrin interacts with the plasma membrane through the proteins ankyrin and 4.1. Ankyrin couples spectrin to the cytosplasmic domain of the HCO3-/Cl- transporter, an abundant TM protein in erythrocytes. Protein 4.1 couples several spectrin proteins to actin and to the cytoplasmic domain of glycophorin. Through protein 4.1, the cortex is mechanically coupled to both the plasma membrane (glycophorin) and to the cytoskeleton of the cell (actin).
V. Topology.
Biological Membranes are Structurally and Functionally Asymmetric. Biological membranes are asymmetric with respect to lipid composition and TM protein orientation. In the plasma membrane for example, glycolipids, PC and sphingomyelin are present predominantly in the outer leaflet, whereas PI, PE and PS are located predominantly in the inner leaflet. TM proteins are almost all vectorial: their function requires that they be always oriented in one direction with respect to the exterior of the cell (for example pumps, receptors and membrane bound enzymes). This structural asymmetry arises from the topological relationship between the ER and Golgi and the other membranes of the cell. Lipid synthesis and ribosomal TM protein synthesis takes place on the cytosolic surface of the ER. Glycosylation of lipids and TM proteins occurs in the lumen of the ER and in the lumen of the Golgi. Mature lipids and proteins are delivered to the plasma membrane by vesicles. Through the events of vesicle extrusion and fusion, the interior surface of the ER and Golgi membranes are topologically equivalent to the exterior surface of the cell.
Clinical Correlations.