Cell Biology Past Paper PDF - PGCCT03, Aliah University

PGCCT03: Cell Biology MSc Botany/Zoology/Microbiology Semester I Dr Safdar Ali Department of Biological Sciences Aliah University Fluid Mosaic Model of Plasma Membrane Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H....

PGCCT03: Cell Biology MSc Botany/Zoology/Microbiology Semester I Dr Safdar Ali Department of Biological Sciences Aliah University Fluid Mosaic Model of Plasma Membrane Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 What does this tell about membrane? Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Electron micrograph of a thin section through an erythrocyte membrane stained with osmium tetroxide. The characteristic “railroad track” appearance of the membrane indicates the presence of two polar layers, consistent with the bilayer structure of phospholipid membranes. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Other Lipid Arrangements Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Synthetic Bilayer Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Properties of synthetic bilayer First, they are virtually impermeable to water-soluble (hydrophilic) solutes, which do not readily diffuse across the bilayer. These solutes include salts, sugars, and most other small hydrophilic molecules— including water itself. The second property of a bilayer is its stability. The hydrophobic and van der Waals interactions between the fatty acyl chains maintain the integrity of the interior of the bilayer structure. Even though the exterior aqueous environment may vary widely in ionic strength and pH, the bilayer has the strength to retain its characteristic architecture. Third, all synthetic phospholipid bilayers can spontaneously form sealed closed compartments in which the aqueous space on the inside is separated from the exterior environment. Two surfaces of a cellular membrane: cytosolic and exoplasmic Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Reversal of faces of membrane Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 How is their membrane different from others? Chloroplast Nucleus Mitochondria Types of Membrane Lipids Phosphoglycerides Sphingolipids Sterols Phosphoglycerides Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Most phosphoglycerides are derivatives of glycerol 3-phosphate Two esterified fatty acyl chains that constitute the hydrophobic “tail” Polar “head group” esterified to the phosphate. The fatty acids can vary in length and be saturated (no double bonds) or unsaturated (one, two, or three double bonds). On the basis of head group Phosphatidylcholine (PC) Phosphatidylethanolamine(PE) Phosphatidylserine (PS) Phosphatidylinositol (PI). Plasmalogens contain one fatty acyl chain attached to glycerol by an ester linkage and one attached by an ether linkage Sphingolipids Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Sphingolipids are derivatives of sphingosine, an amino alcohol with a long hydrocarbon chain. Various fatty acyl chains are connected to sphingosine by an amide bond. Sphingomyelins (SM): phosphocholine head group, are phospholipids. Other sphingolipids are glycolipids in which a single sugar residue or branched oligosaccharide is attached to the sphingosine backbone. Glucosylcerebroside (GlcCer) has a glucose head group. Sterols Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 The major sterols Animals (cholesterol) Fungi (ergosterol) Plants (stigmasterol) The basic structure of sterols is a four-ring hydrocarbon (yellow). Sterols are amphipathic Single hydroxyl group: polar head group Conjugated ring and short hydrocarbon chain: hydrophobic tail Lateral mobility of lipids 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. These diffusion rates indicate that the bilayer is 100 times more viscous than water— about the same as the viscosity of olive oil. A membrane 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. FRAP Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Cholesterol is important in maintaining the appropriate fluidity of natural membranes, a property that appears to be essential for normal cell growth and reproduction. Cholesterol restricts the random movement of phospholipid head groups at the outer surfaces of the leaflets, but its effect on the movement of long phospholipid tails depends on its concentration. At the cholesterol concentrations normally present in the plasma membrane, the interaction of the steroid ring with the long hydrophobic tails of phospholipids tends to immobilize those lipids and thus decreases biomembrane fluidity. It is this property that can help organize the plasma membrane into discrete subdomains of unique lipid and protein composition. At lower cholesterol concentrations, however, the steroid ring separates and disperses phospholipid tails, causing the inner regions of the membrane to become slightly more fluid Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Lipid composition: Bilayer thickness & Curvature Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Although most phospholipids are present in both membrane leaflets, some are commonly more abundant in one or the other leaflet. For instance, in plasma membranes from human erythrocytes and Madin-Darby canine kidney (MDCK) cells grown in culture, almost all the sphingomyelin and phosphatidylcholine, both of which form LESS FLUID bilayers, are found in the exoplasmic leaflet. In contrast, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, which form MORE FLUID bilayers, are preferentially located in the cytosolic leaflet. Phosphatidylserine: Normally abundant in cytosolic leaflet of plasma membrane Platelet stimulation by serum: phosphatidylserine is briefly translocated to the exoplasmic face, presumably by a flippase enzyme, where it activates enzymes participating in blood clotting. When cells die: Phosphatidylserine is increasingly found in the exoplasmic one. [This increased exposure is detected experimentally by use of a labeled version of annexin V, a protein that specifically binds to phosphatidylserine, to measure the onset of programmed cell death (apoptosis)]. Increased exposure of phosphatidylserine on dying or dead cells is recognized by phagocytic cells, which initiate engulfment of such apoptotic bodies and thus ensure timely and safe disposal of cell remnants. Lipid rafts Lipids may be organized within the leaflets Discovery: Lipids remaining after the extraction (solubilization) of plasma membranes with non-ionic detergents such as Triton X-100 predominantly contain two species: cholesterol and sphingomyelin. Because these two lipids are found in more ordered, less fluid bilayers, researchers hypothesized that they form microdomains, termed lipid rafts, surrounded by other, more fluid phospholipids that are more readily extracted by non-ionic detergents. Lipid rafts are typically 50 nm in diameter. Rafts can be disrupted by Methyl-β-cyclodextrin: specifically extracts cholesterol from membranes Antibiotics such as filipin that sequester cholesterol into aggregates They contain a subset of plasma-membrane proteins Sensing extracellular signals and transmitting them into the cytosol Raft fractions are enriched in glycolipids Brings many key proteins into close proximity and stabilizing their interactions Raft-associated glycolipids permits interactions of their tails across the hydrophobic core and help organize lipids of the cytosolic leaflet in the formation of signaling platforms. Lipid droplets Lipid droplets are vesicular structures, composed of triglycerides and cholesterol esters, that originate from the ER and serve a lipid-storage function. When a cell’s supply of lipids exceeds the immediate need for membrane construction, excess lipids are relegated to these lipid droplets [visualized in live cells by staining with a lipophilic dye such as Congo red. Feeding cells with oleic acid, a type of fatty acid, enhances lipid droplet formation]. Also serve as platforms for storage of proteins targeted for degradation. The biogenesis of lipid droplets starts with delamination of the lipid bilayer of the ER through insertion of triglycerides and cholesterol esters The lipid “lens” continues to grow by insertion of more lipid, until finally a lipid droplet is hatched by scission from the ER. The resulting cytoplasmic droplet is thereby surrounded by a phospholipid monolayer. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Membrane Proteins Membrane proteins are defined by their location within or at the surface of a phospholipid bilayer. The proteins associated with a particular membrane are responsible for its distinctive activities. The kinds and amounts of proteins associated with biomembranes vary depending on cell type and subcellular location. Inner mitochondrial membrane is 76 percent protein Myelin membrane that surrounds nerve axons, only 18 percent protein WHY Membrane Proteins Classification On the basis of their position with respect to the membrane Integral Lipid-anchored Peripheral Integral membrane proteins Transmembrane proteins: Span a phospholipid bilayer and comprise three domains Cytosolic [hydrophilic] Exoplasmic [hydrophilic] Membrane-spanning [hydrophobic] One or more α helices Multiple β strands. Glycophorin A Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Bacteriorhodopsin and Glycerol Channel Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 OmpX porins Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Lipid anchored proteins Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Lipid anchored proteins: Acyl anchors Cytosolic proteins such as v-Src are associated with the plasma membrane Through a single fatty acyl chain attached to the N-terminal glycine (Gly) residue of the polypeptide. Myristate (C14) and palmitate (C16) are common acyl anchors. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Lipid anchored proteins: Prenylation Cytosolic Ras and Rab proteins Anchored to the membrane by prenylation of one or two cysteine (Cys) residues at or near the C-terminus. Prenylation: 15-carbon farnesyl or 20-carbon geranylgeranyl group is bound by a thioether bond to the –SH group of a C-terminal cysteine residue of the protein, usually part of a C-terminal Cys-Ala-Ala-X (X = any of a number of amino acids) or CAAX box Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Lipid anchored proteins: GPI Anchor The lipid anchor on the exoplasmic surface of the plasma membrane The phosphatidylinositol part (red) of this anchor contains two fatty acyl chains that extend into the bilayer. The phosphoethanolamine unit (purple) in the anchor links it to the protein. The two green hexagons represent sugar units, which vary in number, nature, and arrangement in different GPI anchors. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Peripheral proteins Do not directly contact the hydrophobic core of the phospholipid bilayer. They are bound to the membrane Indirectly by interactions with integral or lipid-anchored membrane proteins Directly by interactions with lipid head groups. Peripheral proteins can be bound to either the cytosolic or the exoplasmic face of the plasma membrane. Cytoskeletal filaments can be more loosely associated with the cytosolic face, usually through one or more peripheral adapter proteins. Determine the cell’s shape and mechanical properties Communication between the cell interior and the exterior. Peripheral proteins on the outer surface of the plasma membrane and the exoplasmic domains of integral membrane proteins are often attached to components of the extracellular matrix or to the cell walls surrounding bacterial and plant cells, providing a crucial interface between the cell and its environment. Transmembrane Transport Plasma membrane forms the barrier that separates the cytoplasm from the exterior environment, thus defining a cell’s physical and chemical boundaries Maintains essential differences between the composition of the extracellular fluid and that of the cytosol. For example, the concentration of sodium chloride (NaCl) in the blood and extracellular fluids of animals is generally above 150 mM, similar to the ~450 mM Na+ found in the seawater, in which all cells are thought to have evolved. In contrast, the sodium ion (Na+) concentration in the cytosol is tenfold lower, about 15 mM, while the potassium ion (K+) concentration is higher in the cytosol than outside. Few gases and small, uncharged, water-soluble molecules can readily diffuse across a pure phospholipid bilayer. But cellular membranes must serve not only as barriers, but also as conduits, selectively transporting molecules and ions from one side of the membrane to the other. Energy-rich glucose, for example, must be imported into the cell, and wastes must be shipped out. Relative permeability of a pure phospholipid bilayer Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 What factors affect transport across membrane? Gradient Concentration High to low Low to High Electrical Membrane potential Diffusion Gases, such as O2 and CO2, and small uncharged polar molecules, such as urea and ethanol, can readily move across an artificial membrane composed of pure phospholipid or of phospholipid and cholesterol. Such molecules can also diffuse across cellular membranes without the aid of transport proteins. No metabolic energy is expended during simple diffusion because movement is from a high to a low concentration of the molecule, down its chemical concentration gradient. Such movements are spontaneous because they have a positive ΔS value (increase in entropy) and thus a negative ΔG (decrease in free energy). The diffusion rate of any substance across a pure phospholipid bilayer dependent upon Concentration gradient across the bilayer Hydrophobicity Size Hydrophobicity Determined by measuring its partition coefficient K, the equilibrium constant for its partition between oil and water. The higher a substance’s partition coefficient (the greater the fraction found in oil relative to water), the more lipid soluble it is, and therefore, the faster its rate of movement across a bilayer. The first and rate-limiting step in transport by simple diffusion is movement of a molecule from the aqueous solution into the hydrophobic interior of the phospholipid bilayer, which resembles olive oil in its chemical properties. This is the reason that the more hydrophobic a molecule is, the faster it diffuses across a pure phospholipid bilayer. For example, diethylurea, with an ethyl group attached to each nitrogen atom has a K of 0.01 Whereas urea has a K of 0.0002. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Membrane Transport Proteins Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Channels Water, specific ions, or hydrophilic small molecules Movement down their concentration or electric potential gradients. This process requires transport proteins but not energy, facilitated transport. Channels form a hydrophilic “tube” or passageway across the membrane Movement at a very rapid rate. Channels Open much of the time: nongated Open only in response to specific chemical or electrical signals: Gated Channels are very selective for the type of molecule they transport. Transporters Transporters (also called carriers) move a wide variety of ions and molecules Much slower rate than channels Three types Uniporters transport a single type of molecule down its concentration gradient. Collectively, channels and uniporters are sometimes called facilitated transporters, indicating movement down a concentration or electrochemical gradient. Antiporters and Symporters couple the movement of one type of ion or molecule against its concentration gradient with the movement of one or more different ions down its concentration gradient, in the same (symporter) or different (antiporter) directions. These proteins are often called cotransporters because of their ability to transport two or more different solutes simultaneously. ATP Powered Pumps ATP-powered pumps (or simply pumps) are ATPases that use the energy of ATP hydrolysis to move ions or small molecules across a membrane against a chemical concentration gradient, an electric potential, or both. Active transport: coupled chemical reaction. Transport of ions or small molecules “uphill” against an electrochemical gradient, which requires energy, is coupled to the hydrolysis of ATP, which releases energy. ATP pumps use energy from hydrolysis of ATP, whereas cotransporters use the energy stored in an electrochemical gradient. The latter process is sometimes referred to as secondary active transport. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Glucose Transport Uniport vs Simple diffusion 1. The rate of substrate movement by uniporters is far higher than simple diffusion through a pure phospholipid bilayer. 2. Transported molecule never enters the hydrophobic core, partition coefficient K is irrelevant. 3. Transport occurs via a limited number of uniporter molecules. Maximum transport rate, Vmax, achieved when each uniporter is working at its maximal rate. 4. Transport is reversible, and the direction of transport will change if the direction of the concentration gradient changes. 5. Transport is specific. Each uniporter transports only a single type of molecule or a single group of closely related molecules. A measure of the affinity of a transporter for its substrate is the Michaelis constant, Km, which is the concentration of substrate at which transport is half Vmax. GLUT2, with a Km of about 20 mM, has a much lower affinity for glucose than GLUT1, with a Km of about 1.5 mM. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Structural model (side view) of the full-length human GLUT1 protein in an inward- open conformation. The transporter consists of 12 transmembrane α-helical segments.The colors represent the hydrophobicity of the amino acids, with hydrophobic in yellow and hydrophilic in blue. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 A working model for GLUT1. In this alternating access model, the outward-open conformation of GLUT1 binds glucose (step 1 ) and moves to a ligand-bound occluded conformation (step 2 ) before changing to its inward-open conformation (step 3 ) when it delivers glucose to the cytoplasm, then moves through a ligand free occluded conformation (step 4 ) before beginning another round of glucose transport from outside to inside the cell. If the concentration of glucose is higher inside the cell than outside, the cycle will work in reverse (step 4 → step 1 ), resulting in net movement of glucose out of the cell. In addition to glucose, the isomeric sugars d-mannose and d-galactose, which differ from d-glucose in their configuration at only one carbon atom, are transported by GLUT1 at measurable rates. However, the Km for glucose (1.5 mM) is much lower than it is for d-mannose (20 mM) or d-galactose (30 mM). Thus GLUT1 is quite specific, having a much higher affinity (indicated by a lower Km) for its normal substrate d-glucose than for other substrates. GLUT1 accounts for 2 percent of the protein in the plasma membrane of erythrocytes. After glucose is transported into the erythrocyte, it is rapidly phosphorylated, forming glucose-6-phosphate, which cannot leave the cell. Because this reaction, the first step in the metabolism of glucose, is rapid and occurs at a constant rate, the intracellular concentration of glucose is kept low even when glucose is imported from the extracellular environment. Consequently, the concentration gradient of glucose (outside greater than inside the cell) is kept sufficiently high to support continuous, rapid import of additional glucose molecules and provide sufficient glucose for cellular metabolism. The human genome encodes at least 14 highly homologous GLUT proteins, GLUT1– GLUT14, that are all thought to contain 12 membrane-spanning α helices, suggesting that they evolved from a single ancestral transport protein. In the human GLUT1 protein, the transmembrane α helices are predominantly hydrophobic; several helices, however, bear amino acid residues (e.g., serine, threonine, asparagine, and glutamine) whose side chains can form hydrogen bonds with the hydroxyl groups on glucose. Movement of Water Movement of water into and out of cells is an important feature of the life of all organisms. The aquaporins are a family of membrane proteins that allow water and a few other small uncharged molecules, such as glycerol, to cross cellular membranes efficiently. Water spontaneously moves “downhill” across a semipermeable membrane from a solution of lower solute concentration (relatively high water concentration) to one of higher solute concentration (relatively low water concentration), a process termed osmosis, or osmotic flow. Osmosis is equivalent to “diffusion” of water across a semipermeable membrane. Osmotic pressure is defined as the hydrostatic pressure required to stop the net flow of water across a membrane separating solutions of different water concentrations. In other words, osmotic pressure balances the entropy-driven thermodynamic force of the water concentration gradient. In this context, a “membrane” may be a layer of cells or a plasma membrane that is permeable to water but not to the solutes it contains. The osmotic pressure is directly proportional to the difference in the concentrations of the total numbers of solute molecules on the two sides of the membrane. For example, a 0.5 M NaCl solution is actually 0.5 M Na+ ions and 0.5 M Cl− ions and has the same osmotic pressure as a 1 M solution of glucose or sucrose. Osmotic Pressure Solutions A and B are separated by a membrane that is permeable to water but impermeable to all solutes. If CB (the total concentration of solutes in solution B) is greater than CA, water will tend to flow across the membrane from solution A to solution B. Osmotic pressure π is the hydrostatic pressure that would have to be applied to solution B to prevent this water flow. From the van’t Hoff equation, osmotic pressure is given by π = RT(CB − CA), where R is the gas constant and T is the absolute temperature. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 In vascular plants, water and minerals are absorbed from the soil by the roots and move up the plant through conducting tubes (the xylem); water loss from the plant, mainly by evaporation from the leaves, drives this movement of water. Unlike animal cells, plant, algal, fungal, and bacterial cells are surrounded by a rigid cell wall, which resists the expansion of the volume of the cell when the intracellular osmotic pressure increases. Without such a wall, animal cells expand when internal osmotic pressure increases; if that pressure rises too much, the cells burst like overinflated balloons. Because of the cell wall, the osmotic influx of water that occurs when plant cells are placed in a hypotonic solution (even pure water) leads to an increase in intracellular pressure, but not in cell volume. Turgor Pressure in Plants In plant cells, the concentration of solutes (e.g., sugars and salts) is usually higher in the vacuole than in the cytosol, which in turn has a higher solute concentration than the extracellular space. The osmotic pressure generated by the entry of water into the cytosol and then into the vacuole, called turgor pressure, pushes the cytosol and the plasma membrane against the resistant cell wall. Plant cells can harness this pressure to help them stand upright and grow. Cell elongation during growth occurs by means of a hormone-induced, localized loosening of a defined region of the cell wall followed by an influx of water into the vacuole, increasing its size and thus the size of the cell. Protozoans Although most protozoans (like animal cells) do not have a rigid cell wall, many contain a contractile vacuole that permits them to avoid osmotic lysis. A contractile vacuole takes up water from the cytosol and, unlike a plant vacuole, periodically discharges its contents through fusion with the plasma membrane. Thus even though water continuously enters the protozoan cell by osmotic flow, the contractile vacuole prevents too much water from accumulating in the cell and swelling it to the bursting point. Aquaporins Increase the water permeability of cell membranes Frog Oocytes Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 In its functional form, an aquaporin is a tetramer of identical 28-kDa subunits. Each subunit contains six membrane-spanning α helices that form a central pore through which water can move in either direction, depending on the osmotic gradient. The~2-nm-long water-selective channel, or pore, at the center of each monomer is only 0.28 nm in diameter—only slightly larger than the diameter of a water molecule. The molecular sieving properties of the channel are determined by several conserved hydrophilic amino acid residues whose side-chain and carbonyl groups extend into the middle of the channel and by a relatively hydrophobic wall that lines one side of the channel. Several water molecules can move simultaneously through the channel, each molecule sequentially forming specific hydrogen bonds with the channel-lining amino acids and displacing another water molecule downstream. Aquaporins do not undergo conformational changes during water transport, so they transport water orders of magnitude faster than GLUT1 transports glucose. The formation of hydrogen bonds between the oxygen atom of water and the amino groups of two amino acid side chains ensures that only uncharged water (i.e., H2O, but not H3O+) passes through the channel; the orientations of the water molecules in the channel prevent protons from jumping from one to the next and thus prevent the net movement of protons through the channel. As a consequence, ionic gradients are maintained across membranes even when water is flowing across them through aquaporins. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Mammals express a family of aquaporins; 11 such genes are known in humans. Aquaporin 1 is expressed in abundance in erythrocytes, and the homologous aquaporin 2 is found in the kidney epithelial cells that resorb water from the urine, thus controlling the amount of water in the body. The activity of aquaporin 2 is regulated by vasopressin, also called antidiuretic hormone, in a manner that resembles the regulation of GLUT4 activity in fat and muscle. When the cells are in their resting state and water is being excreted to form urine, aquaporin 2 is sequestered in intracellular vesicle membranes and so is unable to mediate water import into the cell. When the polypeptide hormone vasopressin binds to the cell-surface vasopressin receptor, it activates a signaling pathway using cAMP as the intracellular signal that causes these aquaporin 2–containing vesicles to fuse with the plasma membrane, increasing the rate of water uptake and return to the circulation. Inactivating mutations in either the vasopressin receptor or the aquaporin 2 gene cause diabetes insipidus, a disease marked by excretion of large volumes of dilute urine. This finding demonstrates that the level of aquaporin 2 is rate limiting for water resorption from urine being formed by the kidney. Other members of the aquaporin family transport hydroxyl-containing molecules such as glycerol rather than water. Human aquaporin 3, for instance, transports glycerol. ATP Powered Pumps Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 P-class pumps are composed of two catalytic α subunits, which become phosphorylated as part of the transport cycle. Two β subunits, present in some of these pumps, may regulate transport. V-class and F-class pumps do not form phosphoprotein intermediates, and almost all transport only protons. Their structures are similar and contain similar proteins, but none of their subunits are related to those of P-class pumps. V-class pumps couple ATP hydrolysis to transport of protons against a concentration gradient, whereas F-class pumps normally operate in the reverse direction and use the energy in a proton concentration or voltage gradient to synthesize ATP. All members of the large ABC superfamily of proteins contain two transmembrane (T) domains and two cytosolic ATP-binding (A) domains, which couple ATP hydrolysis to solute movement. These core domains are present as separate subunits in some ABC proteins but are fused into a single polypeptide in other ABC proteins. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Ion gradients across the plasma and intracellular membranes Cells expend considerable energy for this Up to 25 percent of the ATP produced by nerve and kidney cells is used for ion transport, and human erythrocytes consume up to 50 percent of their available ATP for this. Both cases, most of this ATP is used to power the Na+/K+ pump. The resultant Na+ and K+ gradients in neurons are essential for their ability to conduct electrical signals rapidly and efficiently. Certain enzymes required for protein synthesis in all cells require a high K+ concentration and are inhibited by high concentrations of Na+; these enzymes would cease to function without the operation of the Na+/K+ pump. In cells treated with poisons that inhibit the production of ATP (e.g., 2,4-dinitrophenol in aerobic cells), the pumping stops, and the ion concentrations inside the cell gradually approach those of the exterior environment as ions spontaneously move through channels in the plasma membrane down their electrochemical gradients. Eventually the treated cells die. Operational model of the Ca2+ ATPase in the SR membrane of skeletal muscle cells Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 All P-class ATP-powered pumps, regardless of which ion they transport, are phosphorylated on a highly conserved aspartate residue during the transport process. As deduced from cDNA sequences, the catalytic α subunits of all the P-class pumps examined to date have similar amino acid sequences and thus are presumed to have similar arrangements of transmembrane α helices and cytosol-facing A ( actuator), P, and N domain. These findings strongly suggest that all such proteins evolved from a common precursor, although they now transport different ions. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 V-Class H+ ATPases Maintain the Acidity of Lysosomes and Vacuoles All V-class ATPases transport only H+ ions. These proton pumps, present in the membranes of lysosomes, endosomes, and plant vacuoles, function to acidify the lumina of these organelles. The pH of the lysosomal lumen can be measured precisely in live cells by use of particles labeled with a pH-sensitive fluorescent dye. When these particles are added to the extracellular fluid, the cells engulf and internalize them, ultimately transporting them into lysosomes. The lysosomal pH can be calculated from the spectrum of the fluorescence emitted Maintenance of the hundredfold or more proton gradient between the lysosomal lumen (pH ~4.5–5.0) and the cytosol (pH ~7.0) depends on a V-class ATPase and thus on ATP production by the cell. The low lysosomal pH is necessary for optimal function of the many proteases, nucleases, and other hydrolytic enzymes in the lumen; on the other hand, a cytosolic pH of 5 would disrupt the functions of many proteins optimized to act at pH 7 and lead to death of the cell. Pumping of relatively few protons is required to acidify an intracellular vesicle. To understand why, recall that a solution of pH 4 has a H+ ion concentration of 10−4 moles per liter, or 10−7 moles of H+ ions per milliliter. There are 6.02 × 1023 atoms of H per mole (Avogadro’s number), so a milliliter of a pH 4 solution contains 6.02 × 1016 H+ ions. Thus at pH 4, a primary spherical lysosome with a volume of 4.18 × 10−15 ml (diameter of 0.2 μm) would contain just 252 protons. At pH 7, the same organelle would have an average of only 0.2 protons in its lumen, and thus pumping of only about 250 protons would be necessary for lysosome acidification. By themselves, V-class proton pumps cannot acidify the lumen of an organelle (or the extracellular space) because these pumps are electrogenic; that is, a net movement of electric charge occurs during transport. Pumping of just a few protons causes a buildup of positively charged H+ ions on the exoplasmic (inside) face of the organelle membrane. For each H+ pumped across, a negative ion (e.g., OH− or Cl−) will be “left behind” on the cytosolic face, causing a buildup of negatively charged ions there. These oppositely charged ions attract each other on opposite faces of the membrane, generating a charge separation, or electric potential, across the membrane. The lysosome membrane thus functions as a capacitor in an electric circuit, storing opposing charges (anions and cations) on opposite sides of a barrier impermeable to the movement of charged particles. As more and more protons are pumped and build up excess positive charge on the exoplasmic face, the energy required to move additional protons against this rising electric potential gradient increases dramatically and prevents pumping of additional protons long before a significant transmembrane H+ concentration gradient is established. In order for an organelle lumen or an extracellular space (e.g., the lumen of the stomach) to become acidic, movement of protons must be accompanied either by (1) movement of an equal number of anions (e.g., Cl−) in the same direction Occurs in lysosomes and plant vacuoles, whose membranes contain V-class H+ ATPases and anion channels through which accompanying Cl− ions or (2) movement of equal numbers of a different cation in the opposite direction. Occurs in the lining of the stomach, which contains a P-class H+/K+ ATPase that is not electrogenic and pumps one H+ outward and one K+ inward Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 ABC superfamily of membrane transport proteins ABC Proteins Export a Wide Variety of Drugs and Toxins from the Cell All members of the very large and diverse ABC superfamily of membrane transport proteins contain two transmembrane (T) domains and two cytosolic ATP-binding (A) domains. The T domains, each built of 10 membrane-spanning α helices, form the pathway through which the transported substance (substrate) crosses the membrane and determine the substrate specificity of each ABC protein. The sequences of the A domains are approximately 30–40 percent homologous in all members of this superfamily, indicating a common evolutionary origin. The first eukaryotic ABC protein to be recognized was discovered during studies on tumor cells and cultured cells that exhibited resistance to several drugs with unrelated chemical structures. Such cells were eventually shown to express elevated levels of a multidrug-resistance (MDR) transport protein originally called MDR1 and now known as ABCB1. This protein uses the energy derived from ATP hydrolysis to export a large variety of drugs from the cytosol to the extracellular medium. The Mdr1 gene is frequently amplified in multidrug-resistant cells, resulting in a large overproduction of the MDR1 protein. In contrast to bacterial ABC proteins, which are built of four discrete subunits, all four domains of mammalian ABCB1 are fused into a single 170-kDa protein. The multidrug transporter ABCB1 (MDR1): Structure and model of ligand export Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 An in vitro fluorescence-quenching assay revealed the phospholipid flippase activity of ABCB4 Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Structure and function of the cystic fibrosis transmembrane regulator (CFTR) Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Ion Channels and Membrane Potential Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 If [Naright]/[Naleft] = 10, a tenfold ratio of concentrations, then ENa = +0.059 V (or +59 mV) The Resting Membrane Potential in Animal Cells Depends Largely on the Outward Flow of K+ Ions Through Open K+ Channels The plasma membranes of animal cells contain many open K+ channels but few open Na+, Cl−, or Ca2+ channels. As a result, the major ionic movement across the plasma membrane s the movement of K+ from the inside outward, powered by the K+ concentration gradient. This movement leaves an excess of negative charge on the cytosolic face of the plasma membrane and creates an excess of positive charge on the exoplasmic face This outward flow of K+ ions is the major determinant of the inside-negative membrane potential. The channels through which the K+ ions flow, called resting K+ channels, alternate, like all channels, between an open and a closed state, but since their opening and closing is not affected by the membrane potential or by small signaling molecules, these channels are referred to as nongated. Quantitatively, the usual resting membrane potential of –60 to –70 mV is close to the potassium equilibrium potential, calculated from the Nernst equation. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Structure of a resting K+ channel from the bacterium Streptomyces lividans. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Backbone carbonyl oxygens on residues located in a Gly-Tyr-Gly sequence that is found in an analogous position in the P segment Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Two Na+/One-glucose symporter Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Three-Na+/ One-Ca2+ antiporter A Na+-Linked Ca2+ Antiporter Regulates the Strength of Cardiac Muscle Contraction Carbon dioxide transport in blood: Cl−/HCO3− antiporter Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Concentration of ions and sucrose by the plant vacuole. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Transcellular Transport Absorption of many nutrients from the intestinal lumen across the epithelial cell layer and eventually into the blood occurs by a Two-stage process called transcellular transport: import of molecules through the plasma membrane on the apical side of intestinal epithelial cells and their export through the plasma membrane on the basolateral (blood-facing) side Transcellular transport of glucose from the intestinal lumen into the blood Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016 Acidification of the stomach lumen by parietal cells in the gastric lining. Molecular Cell Biology, 8th edition Harvey Lodish et al, New York: W. H. Freeman; 2016