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14 The Plasma Membrane 14 The Plasma Membrane • Structure of the Plasma Membrane • Transport of Small Molecules • Endocytosis Introduction All cells are surrounded by a plasma membrane. The plasma membrane: • Defines the cell boundary and separates it from the environment. • Is a selective barrier, and determines the composition of the cytoplasm. • Mediates interactions between the cell and its environment. Structure of the Plasma Membrane The fundamental structure of the membrane is the phospholipid bilayer. Proteins embedded in the bilayer carry out specific functions, including selective transport of molecules and cell-cell recognition. Structure of the Plasma Membrane Mammalian red blood cells (erythrocytes) have been useful as a model for studies of membrane structure. These cells have no nuclei or internal membranes, making it easy to isolate pure plasma membranes. Structure of the Plasma Membrane The bilayer structure can be seen in electron micrographs. The polar head groups appear as dark lines because they bind the electrondense metal stains. The hydrophobic fatty acid chains in the center are lightly stained. Figure 14.1 Bilayer structure of the plasma membrane Structure of the Plasma Membrane Animal cell plasma membranes have five major phospholipids: • Outer leaflet—phosphatidylcholine and sphingomyelin. • Inner leaflet— phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol Table 14.1 Lipid Composition of the Plasma Membrane Figure 14.2 Lipid components of the plasma membrane Structure of the Plasma Membrane Animal cell plasma membranes also contain: Glycolipids—only in outer leaflet, with carbohydrate portions exposed on the cell surface. Cholesterol—present in about the same molar amounts as phospholipids. Structure of the Plasma Membrane Phospholipid structure is responsible for the basic function of membranes— separating aqueous compartments. The bilayer interior consists of hydrophobic fatty acid chains, so it is impermeable to water-soluble molecules—ions and most biological molecules. Structure of the Plasma Membrane Bilayers are viscous fluids, not solid. The fatty acids have one or more double bonds, which make kinks in the chain and keep them from packing together. Lipids and proteins are free to diffuse laterally within the membrane. Structure of the Plasma Membrane Cholesterol affects membrane fluidity and is involved in formation of functional domains in the membrane. Cholesterol and the sphingolipids (sphingomyelin and glycolipids) tend to cluster in small semisolid patches called lipid rafts. Figure 14.3 Lipid rafts Structure of the Plasma Membrane Most plasma membranes are about 50% lipid and 50% protein by weight. Since proteins are much larger than lipids, this corresponds to about one protein per 50–100 molecules of lipid. Structure of the Plasma Membrane The fluid mosaic model of membrane structure was proposed by Singer and Nicolson in 1972: Membranes are two-dimensional fluids with proteins inserted into lipid bilayers. Both proteins and lipids are able to diffuse laterally through the membrane. Figure 14.4 Fluid mosaic model of the plasma membrane Structure of the Plasma Membrane Lateral movement of proteins and lipids was first demonstrated in 1970. Human and mouse cells were fused in culture, then analyzed for membrane proteins using fluorescent antibodies. Within 40 minutes, the mouse and human proteins became intermixed over the surface of hybrid cells. Figure 14.5 Mobility of membrane proteins Structure of the Plasma Membrane Peripheral membrane proteins: associated with membranes through protein-protein interactions; often ionic bonds. The bonds can be disrupted by polar reagents (salts or extreme pH). Many are part of the cortical cytoskeleton: spectrin, actin, band 4.1, etc. Structure of the Plasma Membrane Integral membrane proteins: inserted into the lipid bilayer; they can be dissociated only by reagents that disrupt hydrophobic interactions. Detergents are amphipathic molecules with hydrophobic and hydrophilic groups that can solubilize these proteins. Figure 14.6 Solubilization of integral membrane proteins by detergents Structure of the Plasma Membrane Transmembrane proteins: integral proteins that span the lipid bilayer with portions exposed on both sides. They can be seen in electron micrographs of plasma membranes prepared by freeze-fracture technique. Figure 14.7 Freeze-fracture electron micrograph of human red blood cell membranes Structure of the Plasma Membrane The membrane-spanning portions are usually α helices of hydrophobic amino acids; they are inserted into the ER membrane during synthesis. Carbohydrate groups are added in the ER and Golgi; most are glycoproteins with oligosaccharides exposed on the cell surface. Structure of the Plasma Membrane Glycophorin and band 3 illustrate transmembrane protein structure. Glycophorin has a single transmembrane α helix. Band 3 is the transporter for HCO3 – and Cl– ions, with 14 transmembrane α helices. Figure 14.8 Integral membrane proteins of red blood cells Structure of the Plasma Membrane The first transmembrane protein to be analyzed by X-ray crystallography was the photosynthetic reaction center of the bacterium Rhodopseudomonas viridis. Figure 14.9 A bacterial photosynthetic reaction center Structure of the Plasma Membrane Some proteins are anchored in the plasma membrane by covalently attached lipids or glycolipids. Glycosylphosphatidylinositol (GPI) anchors are added to the C terminus of some proteins in the ER. These proteins are glycosylated and exposed on the cell surface. Figure 14.10 Examples of proteins anchored in the plasma membrane by lipids and glycolipids Structure of the Plasma Membrane Other proteins are anchored in the inner leaflet by covalently attached lipids. They are translated on free ribosomes and modified by myristic acid, prenyl groups, or palmitic acid. Many of these proteins (including Src and Ras) play roles in signal transmission. Structure of the Plasma Membrane Glycocalyx: carbohydrate coat formed by the oligosaccharides of glycolipids and glycoproteins. Protects the cell surface from ionic and mechanical stress and forms a barrier to invading microorganisms. Oligosaccharides of the glycocalyx participate in a variety of cell–cell interactions. Figure 14.11 The glycocalyx Structure of the Plasma Membrane Ammendments to the fluid mosaic model: • Mobility of many plasma membrane proteins is restricted. • Membranes are composed of distinct domains that have different structural and functional roles. Structure of the Plasma Membrane Many epithelial cells are polarized; plasma membranes are divided into apical and basolateral domains. In the small intestine, the apical surface is covered by microvilli that increase surface area for absorption. The basolateral surface mediates transfer of absorbed nutrients to the blood. Figure 14.12 A polarized intestinal epithelial cell Structure of the Plasma Membrane To maintain these functions, mobility of plasma membrane proteins must be restricted to appropriate domains. Tight junctions separate the apical and basolateral domains. Membrane proteins can move within each domain but can’t cross from one to the other. Structure of the Plasma Membrane Mobility of many plasma membrane proteins is restricted by association with the cytoskeleton or specialized lipid domains. Transmembrane proteins anchored to the cytoskeleton have restricted mobility and may also act as barriers that limit mobility of other membrane proteins. Figure 14.13 Updated fluid mosaic model Structure of the Plasma Membrane Lipid rafts are transient structures in which specific proteins can be concentrated to facilitate interactions. They are enriched in GPI-anchored proteins and transmembrane proteins involved in a variety of functions, including cell signaling, cell movement, and endocytosis. Figure 14.14 Super-resolution microscopy of lipid rafts Structure of the Plasma Membrane Caveolae are small lipid rafts that start as invaginations of the plasma membrane, organized by caveolin. They have been implicated in endocytosis, cell signaling, regulation of lipid transport, and protection of the plasma membrane against mechanical stress. Figure 14.15 Caveolae Transport of Small Molecules Plasma membranes are selectively permeable to small molecules. Specific transport and channel proteins mediate passage of glucose, amino acids, and other small molecules and ions. Transport of Small Molecules Facilitated diffusion: Direction of movement determined by concentration gradients; no energy required. Transport is mediated by proteins, which allow polar and charged molecules to cross the plasma membrane (carbohydrates, amino acids, ions, nucleosides). Transport of Small Molecules Carrier proteins bind molecules on one side of the membrane, then undergo conformational changes that allow the molecule to pass through and be released on the other side. Transport of Small Molecules Channel proteins form open pores through the membrane, allowing free diffusion of any molecule of the appropriate size and charge. Transport of Small Molecules Carrier proteins allow facilitated diffusion of sugars, amino acids, and nucleosides. The glucose transporter has 12 α-helical transmembrane segments (typical of many carrier proteins). Transport of Small Molecules Glucose transporters function by alternating between two conformational states. A glucose-binding site is alternately exposed on the outside and the inside of the cell. Figure 14.16 Facilitated diffusion of glucose (Part 1) Figure 14.16 Facilitated diffusion of glucose (Part 2) Transport of Small Molecules Glucose is rapidly metabolized in the cell, so intracellular glucose concentrations remain low and glucose is transported into the cell. Glucose transport can also be reversed, (e.g. in liver cells when glucose is synthesized and released into the circulation). Transport of Small Molecules Channel proteins, such as porins, form open pores in the membrane that allow molecules to pass freely. Aquaporins allow water molecules to cross the membrane rapidly. They are impermeable to charged ions, allowing passage of water without affecting electrochemical gradients. Figure 14.17 Structure of an aquaporin Transport of Small Molecules Ion channels are well studied in nerve and muscle cells, where their opening and closing is responsible for transmission of electric signals. Transport through ion channels is extremely rapid: more than a million ions per second. Transport of Small Molecules Ion channels are highly selective; specific channel proteins allow passage of Na+, K+, Ca2+, and Cl–. Most have “gates” that open only in response to specific stimuli. Figure 14.18 Model of an ion channel Transport of Small Molecules Ligand-gated channels open in response to binding of neurotransmitters or other signaling molecules. Voltage-gated channels open in response to changes in electric potential across the plasma membrane. Transport of Small Molecules The role of ion channels in transmitting electric impulses was first shown using giant squid axons by Hodgkin and Huxley in 1952. Electrodes inserted in the axon measured changes in membrane potential resulting from opening and closing of Na+ and K+ channels. Transport of Small Molecules Ion pumps use energy from ATP hydrolysis to actively transport ions across the plasma membrane to maintain concentration gradients. Thus, the ionic composition of the cytoplasm is substantially different from that of extracellular fluids. Table 14.2 Intracellular and Extracellular Ion Concentrations Transport of Small Molecules Because ions are electrically charged, pumping results in electric gradients across the plasma membrane. In resting squid axons, there is an electric potential of about 60 mV; the inside of the cell is negative with respect to the outside. Figure 14.19 Ion gradients and resting membrane potential of the giant squid axon Transport of Small Molecules Na+ is pumped out of the cell while K+ is pumped in. The plasma membrane also contains open K+ channels, so the flow of K+ makes the largest contribution to resting membrane potential. Transport of Small Molecules The Nernst equation describes the relationship between ion concentration and membrane potential: RT Co V ln zF Ci Transport of Small Molecules RT Co V ln zF Ci V—equilibrium potential in volts R—gas constant T—absolute temperature Z—charge of the ion F—Faraday’s constant Co and Ci —concentrations of the ion outside and inside the cell Transport of Small Molecules As nerve impulses (action potentials) travel along axons, the membrane depolarizes. Membrane potential goes from –60 mV to +30 mV in less than a millisecond. This results from rapid sequential opening and closing of voltage-gated Na+ and K+ channels. Figure 14.20 Membrane potential and ion channels during an action potential (Part 1) Figure 14.20 Membrane potential and ion channels during an action potential (Part 2) Transport of Small Molecules Depolarization of adjacent regions of the plasma membrane allows action potentials to travel the length of a nerve cell. At the nerve end, neurotransmitters are released into the synapse where they bind to receptors on another nerve cell to open ligand-gated ion channels. Figure 14.21 Signaling by neurotransmitter release at a synapse Transport of Small Molecules Nicotinic acetylcholine receptors in muscle cells are ligand-gated channels: Binding of acetylcholine opens a channel that allows rapid influx of Na+, which depolarizes the cell membrane and triggers an action potential. Binding of acetylcholine induces a conformational change in the receptor. Figure 14.22 Model of the nicotinic acetylcholine receptor Transport of Small Molecules Voltage-gated Na+ and K+ channels are more selective. Na+ (0.95 Å) is smaller than K+ (1.33 Å), and it is thought that the Na+ channel pore is too narrow for K+ or larger ions. Figure 14.23 Ion selectivity of Na+ channels Transport of Small Molecules The structure of K+ channels was determined by X-ray crystallography. Part of the channel is lined with carbonyl oxygen (C=O) atoms from the polypeptide backbone. They displace the water to which K+ is bound, and the K+ ion passes through. Na+ is too small to interact and remains bound to water. Figure 14.24 Selectivity of K+ channels Transport of Small Molecules Voltage-gated Na+, K+, and Ca2+ channels belong to a family of related proteins. Ion channels play critical roles in signaling in all cell types. Regulated opening and closing of ion channels is a sensitive and versatile mechanism for responding to environmental stimuli. Figure 14.25 Structures of voltage-gated cation channels (Part 1) Figure 14.25 Structures of voltage-gated cation channels (Part 2) Figure 14.25 Structures of voltage-gated cation channels (Part 3) Transport of Small Molecules In active transport, molecules are transported against their concentration gradients. Energy is provided by a coupled reaction (such as ATP hydrolysis). Transport of Small Molecules Ion pumps are examples of active transport. The Na+-K+ pump (Na+-K+ ATPase) uses energy from ATP hydrolysis to transport Na+ and K+ against their electrochemical gradients. Figure 14.26 Structure of the Na+–K+ pump Transport of Small Molecules The Na+-K+ pump operates by ATPdriven conformational changes. 3 Na+ are transported out of the cell and 2 K+ are transported into the cell for every ATP used. Figure 14.27 Model for operation of the Na+–K+ pump (Part 1) Figure 14.27 Model for operation of the Na+–K+ pump (Part 2) Transport of Small Molecules The Na+-K+ pump uses nearly 25% of the ATP in many animal cells. The gradients are necessary for propagation of electric signals in nerve and muscle cells, to drive active transport of other molecules, and to maintain osmotic balance and cell volume. Figure 14.28 Ion gradients across the plasma membrane of a typical mammalian cell Transport of Small Molecules The differences in ion concentrations balance the high concentrations of organic molecules inside cells, equalizing osmotic pressure and preventing the net influx of water. Transport of Small Molecules The Ca2+ pump is also powered by ATP hydrolysis. Ca2+ is transported out of the cell or into the ER lumen, so intracellular Ca2+ concentrations are extremely low. Transient, localized increases in intracellular Ca2+ are important in cell signaling (as in muscle contraction). Figure 14.29 Structure of the Ca2+ pump Transport of Small Molecules Ion pumps in bacteria, yeasts, and plant cells transport H+ out of the cell. H+ is pumped out of stomach lining cells, resulting in the acidity of gastric fluids. Structurally distinct pumps transport H+ into lysosomes and endosomes. Transport of Small Molecules ATP synthases of mitochondria and chloroplasts are another type of H+ pump. These pumps operate in reverse, with the movement of ions down the electrochemical gradient used to drive ATP synthesis. Transport of Small Molecules ABC transporters have highly conserved ATP-binding domains or ATP-binding cassettes. More than 100 members of this family have been identified in both prokaryotic and eukaryotic cells. Transport of Small Molecules All ABC transporters use energy from ATP hydrolysis to transport molecules in one direction. In prokaryotes, they transport nutrient molecules into the cell. In both prokaryotic and eukaryotic cells they transport toxic substances out of the cell. Transport of Small Molecules ABC transporters have two ATP-binding domains and two transmembrane domains. The substrate binding site alternates between outward facing and inward facing, depending on ATP binding and hydrolysis. Figure 14.30 Model of active transport by an ABC transporter Transport of Small Molecules The first eukaryote ABC transporter was discovered as a product of the mdr (multidrug resistance) gene. MDR transporters remove potentially toxic foreign compounds from cells. They are often expressed at high levels in cancer cells, and can remove a variety of chemotherapy drugs. Transport of Small Molecules In cystic fibrosis, defective Cl– transport in epithelial cells results in abnormally thick, sticky mucus which obstructs respiratory passages. The cystic fibrosis gene encodes a protein (CFTR or cystic fibrosis transmembrane conductance regulator) in the ABC transporter family. Transport of Small Molecules Cystic fibrosis is the result of a mutation in CFTR that interferes with proper folding of the protein. Isolation of the gene allows for potential genetic screening and gene therapy. Drugs such as ivacaftor, which increases Cl– transport, are being developed. Molecular Medicine, Ch. 14, p. 557 Transport of Small Molecules Some molecules can be transported against their concentration gradients using energy from coupled transport of another molecule in the energetically favorable direction. Gradients established by Na+-K+ and H+ pumps provide a source of energy for active transport. Transport of Small Molecules Glucose transporters in the apical domain of intestine epithelial cells transport two Na+ and one glucose into the cell. Flow of Na+ down its electrochemical gradient provides the energy that drives uptake of glucose against its concentration gradient. Figure 14.31 Active transport of glucose Transport of Small Molecules In the basolateral domain, glucose is transferred to the underlying connective tissue and blood capillaries by facilitated diffusion. The system is driven by Na+-K+ pumps. Figure 14.32 Glucose transport by intestinal epithelial cells Transport of Small Molecules Uptake of glucose and Na+ is an example of symport—transport of two molecules in the same direction. Facilitated diffusion of glucose is an example of uniport—transport of a single molecule. Transport of Small Molecules Antiport—two molecules are transported in opposite directions. Ca2+ is exported from cells by the Ca2+ pump and by a Na+-Ca2+ antiporter that transports Na+ in and Ca2+ out. Na+-H+ antiporter transports Na+ into the cell and H+ out, preventing acidification by H+ produced in metabolism. Figure 14.33 Examples of antiport Endocytosis Endocytosis allows cells to take up macromolecules, fluids, and large particles such as bacteria. The material is surrounded by an area of plasma membrane, which buds off inside the cell to form a vesicle containing the ingested material. Endocytosis Phagocytosis (cell eating) occurs in specialized cell types. Binding of a particle to receptors on the cell surface triggers extension of pseudopodia, which surround the particle and fuse to form a large vesicle called a phagosome. Endocytosis Phagosomes fuse with lysosomes to form phagolysosomes, in which the material is digested by acid hydrolases. Figure 14.34 Phagocytosis Endocytosis Many amoebas use phagocytosis to capture food particles, such as bacteria. In multicellular animals, phagocytosis is used as a defense against invading microorganisms, and to eliminate aged or damaged cells. Figure 14.35 Examples of phagocytic cells Endocytosis In mammals, macrophages and neutrophils (white blood cells) are the “professional phagocytes.” They remove microorganisms from infected tissues, and macrophages eliminate aged or dead cells from tissues throughout the body. Endocytosis Macropinocytosis: uptake of extracellular fluids in large vesicles. Lamellipodia (sheet-like projections of the plasma membrane) curve into open cups, followed by membrane fusion to form a large intracellular vesicle. Endocytosis Clathrin-mediated endocytosis is a mechanism for selective uptake of specific macromolecules. Mechanisms of cargo selection, vesicle budding, and vesicle fusion are similar to those involved in vesicular transport in the secretory pathway. Endocytosis Macromolecules bind to cell surface receptors in specialized regions called clathrin-coated pits. The pits bud from the membrane with the help of dynamin, to form small clathrin-coated vesicles; these then fuse with early endosomes. Figure 14.36 Clathrin-coated vesicle formation (Part 1) Figure 14.36 Clathrin-coated vesicle formation (Part 2) Endocytosis Clathrin-mediated endocytosis was first studied in patients with familial hypercholesterolemia (FH). Cholesterol is transported through the bloodstream mostly in the form of lowdensity lipoprotein, or LDL particles. Uptake of LDL requires binding to specific receptors in clathrin-coated pits. Figure 14.37 Structure of LDL Endocytosis LDL binding sites on normal cells were determined by adding radiolabeled LDL to cell cultures. Cells of FH patients did not bind LDL. Mutations in the LDL receptors prevent them from binding LDL, or prevent the receptors from concentrating in the coated pits. Key Experiment, Ch. 14, p. 564 (3) Endocytosis Mutations that prevent LDL receptors from concentrating in coated pits are in the internalization signal in the cytoplasmic tail of the receptor. The signal is a sequence of six amino acids, including tyrosine. Similar signals are found in other receptors taken up via clathrin-coated pits. Figure 14.38 The LDL receptor Endocytosis After internalization, clathrin-coated vesicles shed their coats and fuse with early endosomes. The molecules are sorted, recycled to the plasma membrane, or remain in the early endosomes as they mature to late endosomes and lysosomes for degradation. Figure 14.39 Sorting in early endosomes Endocytosis Early endosomes have membrane H+ pumps which maintain acidic internal pH (6.0 to 6.2). This causes dissociation of many ligands from their receptors. Receptors can be returned to the plasma membrane via transport vesicles. Ligands such as LDL remain and are degraded to release cholesterol. Endocytosis About 50% of the plasma membrane is internalized by receptor-mediated endocytosis every hour and must be replaced at an equivalent rate by recycling. Most of this internalized membrane is replaced by recycling. Endocytosis Clathrin-independent endocytosis does not involve specific membrane receptors or coated vesicles. Macropinocytosis and internalization of caveolae are examples. One pathway mediates uptake of GPIanchored plasma membrane proteins clustered in lipid rafts.