Once a transport vesicle has recognized its target membrane and docked there, the vesicle has to
47 Once a transport vesicle has recognized its target membrane and docked there, the vesicle has to
unload its cargo by membrane fusion. Membrane fusion does not always follow immediately, however. As we have seen, in regulated exocytosis fusion does not occur until it is triggered by an extracellular signal.
Docking and fusion are two distinct and separable processes. It is possible, for example, to prevent fusion while permitting docking by keeping the cytosolic concentration of Ca 2+ very low. This results in an accumulation of vesicles attached to but not fused with their target membrane. Docking requires only that the two membranes come close enough for proteins protruding from the lipid bilayers to interact and adhere. Fusion requires a much closer approach, bringing the lipid bilayers to within 1.5 nm of each other so that they can join. For this close approach water must be displaced from the hydrophilic surface of the membranea process that is energetically highly unfavorable. It seems likely that all membrane fusions in cells are catalyzed by specialized fusion proteins that provide a way to cross this energy barrier. The mechanism is still poorly understood. In the case of coatomer-coated transport vesicles at least, fusion with the target membrane requires ATP, GTP, acyl CoA, and several protein components. Two known essential protein components, called NSF and SNAPs (for reasons explained in the legend to Figure 13-58), cycle between the membranes to be fused and the cytosol. The SNAPs bind to both v-SNARE on the vesicle membrane and t-SNARE on the target membrane to initiate the assembly of the fusion apparatus, which catalyzes the fusion of the two lipid bilayers at the vesicle-target-membrane interface (Figure 13-58).
Th e B e s t - c h a ra c t e riz e d Me m b ra n e - Fu s io n P ro t e in I s Ma d e b y a
Viru s 48
Membrane fusion is important in other processes besides vesicular transport, and in particular the simpler membrane fusions that are catalyzed by viral fusion proteins are understood in some detail. Viral fusion proteins play a crucial part in permitting the entry of enveloped viruses (which have a lipid- bilayer-based membrane coat) into the cells that they infect (discussed in Chapter 6). Viruses such as the influenza virus, for example, enter the cell by receptor-mediated endocytosis and are delivered to Membrane fusion is important in other processes besides vesicular transport, and in particular the simpler membrane fusions that are catalyzed by viral fusion proteins are understood in some detail. Viral fusion proteins play a crucial part in permitting the entry of enveloped viruses (which have a lipid- bilayer-based membrane coat) into the cells that they infect (discussed in Chapter 6). Viruses such as the influenza virus, for example, enter the cell by receptor-mediated endocytosis and are delivered to
The genes encoding several viral fusion proteins have been cloned and used to transfect eucaryotic cells in culture. These transfected cells express the viral proteins on their surface, and under appropriate conditions they fuse to form giant multinucleated cells. In the best-studied case, that of the influenza virus, the three-dimensional structure of the fusion protein has been determined by x-ray crystallography. It has been shown that low pH induces a large conformational change in the fusion protein, exposing a previously buried hydrophobic region on the surface of the protein that can interact with the lipid bilayer of a target membrane. A cluster of such hydrophobic regions on closely spaced fusion-protein molecules is thought to bring the two lipid bilayers into close apposition and to destabilize them so that the bilayers fuse (Figure 13-60).
Recently, a mammalian fusion protein has been identified that resembles viral fusion proteins, and it is thought to mediate the fusion of the plasma membranes of sperm and egg that occurs at fertilization (discussed in Chapter 20). As all of these examples emphasize, under normal circumstances membranes do not fuse easily. Membrane fusion requires special proteins and is subject to highly selective controls - a constraint that is crucial both for maintaining the identity of the cell itself and for maintaining the individuality of each of the intracellular compartments.
S u m m a ry
The differences between the membranous compartments of a cell are maintained by an input of free energy, driving directed, selective transport of particular membrane components from one compartment to another. Transport vesicles bud from specialized coated regions of the donor membrane. The assembly of the coat helps to drive the formation of the vesicle. There are two well- characterized types of coated vesicles: clathrin-coated vesicles mediate selective vesicular transport from the plasma membrane and the trans Golgi network, while coatomer-coated vesicles mediate non- selective vesicular transport from the ER and Golgi cisternae. Adaptins provide a molecular link between clathrin coats and specific membrane receptors and thereby mediate the selective uptake of cargo molecules into clathrin-coated vesicles. Coated vesicles have to lose their coat to fuse with their appropriate target membrane in the cell: clathrin coats are lost soon after the vesicle pinches off from the donor membrane, whereas coatomer coats are lost after the vesicle has docked on the target membrane.
Several classes of monomeric GTPases, including ARF and the Rab proteins, help regulate various steps in vesicular transport, including vesicle budding, docking, and fusion. ARF, Rab, and v-SNARE proteins are incorporated during budding into the transport vesicles and help ensure that the vesicles deliver their contents only to the appropriate membrane-bounded compartment: ARF is thought to mediate coatomer (and probably clathrin) coat assembly and coatomer coat disassembly, while Rab proteins are thought to help ensure the specificity of vesicle docking by locking the vesicle onto the target membrane only when complementary vesicle and target membrane snares interact. Membrane fusion is then catalyzed by a number of cytosolic proteins, including SNAPs and NSF, that assemble into a fusion complex at the docking site.
Figure 13-46. Chemical energy is used to give unidirectionality to vesicular transport. In this hypothetical example protein P is an ATP-driven H + pump that is present in low concentration in compartment A and in high concentration in compartment B. Because of the high concentration of P in
compartment B, the lumen of this organelle will be at a much lower pH than that of compartment A. If P undergoes a pH-dependent conformational change that allows it to enter budding vesicles at the higher pH of compartment A but prevents it from doing so at the lower pH of compartment B, then a unidirectional flux of P will occur. As long as the pH difference between the two compartments is maintained through the continuous use of free energy in the form of ATP hydrolysis to drive the H + pump, the concentration gradient of P between the two compartments will be self-sustaining. As discussed in Chapter 12, most membranes are never created de novo but grow by expansion of existing membrane. Thus, although this simple model fails to address how the gradient of P between the two compartments was initially established, it does provide an example of how a cell could use energy to maintain the character of its compartments.
Figure 13-47. Comparison of clathrin-coated and coatomer-coated vesicles. (A) Electron micrograph of clathrin-coated vesicles. (B) Electron micrograph of Golgi cisternae from a cell-free system in which coatomer-coated vesicles bud in the test tube. Note that the clathrin-coated vesicles have a more Figure 13-47. Comparison of clathrin-coated and coatomer-coated vesicles. (A) Electron micrograph of clathrin-coated vesicles. (B) Electron micrograph of Golgi cisternae from a cell-free system in which coatomer-coated vesicles bud in the test tube. Note that the clathrin-coated vesicles have a more
Figure 13-48. Caveolae on the plasma membrane of a human fibroblast. (A) Electron micrograph of a fibroblast in cross-section showing caveolae as deep indentations in the plasma membrane. (B) Deep- etch electron micrograph showing numerous caveolae at the cytoplasmic side of the plasma membrane. Their coat appears to be made of concentrically arranged threads that contain the transmembrane protein caveolin. Note that caveolae differ in both size and structure from clathrin- coated pits, one of which is seen at the top right of (B). (Courtesy of R.G.W. Anderson, from K.G. Rothberg et al., Cell 68:673-682, 1992. © Cell Press.)
Figure 13-49. Clathrin-coated pits and vesicles. This rapid-freeze, deep-etch electron micrograph shows numerous clathrin-coated pits and vesicles on the inner surface of the plasma membrane of cultured fibroblasts. The cells were rapidly frozen in liquid helium, fractured, and deep-etched to Figure 13-49. Clathrin-coated pits and vesicles. This rapid-freeze, deep-etch electron micrograph shows numerous clathrin-coated pits and vesicles on the inner surface of the plasma membrane of cultured fibroblasts. The cells were rapidly frozen in liquid helium, fractured, and deep-etched to
Figure 13-50. The structure of a clathrin coat. (A) Electron micrographs of clathrin triskelions shadowed with platinum. Although this feature cannot be seen in these micrographs, each triskelion is composed of 3 clathrin heavy chains and 3 clathrin light chains. (B) A schematic drawing of the probable arrangement of triskelions on the cytosolic surface of a clathrin-coated vesicle. Two triskelions are shown, with the heavy chains of one in red and of the other in gray; the light chains are shown in yellow. The overlapping arrangement of the flexible triskelion arms provides both mechanical strength and flexibility. Note that the end of each leg of the triskelion turns inward, so that its amino-terminal domain forms an intermediate shell. (C) A three-dimensional reconstruction of a clathrin coat composed of 36 triskelions organized in a network of 12 pentagons and 6 hexagons. The outer, red polygonal shell represents the overlapping legs of the clathrin triskelions; the intermediate, green shell, the amino-terminal domains of the triskelions; and the inner, blue shell, the adaptor proteins that we discuss later. Although the coat shown is too small to enclose a membrane vesicle, the clathrin coats on vesicles are constructed in a similar way from 12 pentagons plus a larger number of hexagons. (A, from E. Ungewickell and D. Branton, Nature 289:420-422, 1981, © 1981 Macmillan Journals Ltd.; B, from I.S. Nathke et al., Cell 68:899-910, 1992. ©Cell Press, from G.P.A. Vigers, R.A. Crowther, and B.M.F. Pearse, EMBO J. 5:2079-2085, 1986.)
Figure 13-51. The assembly and disassembly of a clathrin coat. The assembly of the coat is thought to introduce curvature into the membrane, which leads in turn to the formation of uniformly sized coated buds. The pinching off of the bud to form a vesicle involves the more complex process of membrane fusion, which we discuss later. Although coats consist of multiple protein components, only clathrin is shown in this simplified schematic drawing. Whereas the coat of clathrin-coated vesicles is rapidly removed shortly after the vesicle forms, we shall see later that coatomer coats are removed after the vesicle docks on its target membrane.
Figure 13-52. Selective transport mediated by clathrin-coated vesicles. The adaptins bind both clathrin Figure 13-52. Selective transport mediated by clathrin-coated vesicles. The adaptins bind both clathrin
Figure 13-53. The peptide signal for endocytosis. The various cell-surface receptor proteins that are endocytosed in clathrin-coated vesicles are thought to share this signal, which is recognized by the adaptins that function in receptor-mediated endocytosis from the plasma membrane. The amino acids shown form an essential part of the signal.
Figure 13-54. Selected and nonselected vesicular transport in nonpolarized cells. Nonselected (constitutive) transport (blue arrows) is postulated to be mediated by coatomer-coated vesicles, while various forms of selected (signal-mediated) transport (red arrows) are postulated to be carried out by clathrin-coated vesicles. In polarized cells an additional signaled pathway from the trans Golgi network is required
Figure 13-55. A current model of coatomer-coated vesicle formation. (A) Inactive, soluble ARF-GDP binds to a guanine-nucleotide-releasing protein in the donor membrane, causing the ARF to release its GDP and bind GTP. A GTP-triggered conformational change in ARF exposes its fatty acid chain, which inserts into the donor membrane. (B) Membrane-bound, active ARF-GTP recruits coatomer subunits to the membrane. This causes the membrane to form a bud. A sub-sequent membrane-fusion event pinches off and releases the coated vesicle. The drug brefeldin A blocks coatomer-coat assembly by inhibiting the exchange reaction of GDP to GTP. This blocks coatomer-coated vesicular traffic from the ER through the Golgi apparatus, causing the Golgi apparatus to empty into the ER, as explained on page 604.
Figure 13-56. The postulated role of SNAREs in guiding vesicular transport. Complementary sets of vesicle-SNAREs (v-SNAREs) and target-membrane SNAREs (t-SNAREs) determine the selectivity of transport-vesicle docking. v-SNAREs, which are co-packaged with the coat proteins during the budding of transport vesicles from the donor membrane, bind to complementary t-SNAREs in the target membrane.
Figure 13-57. Postulated role of Rab proteins in ensuring specificity in the docking of transport vesicles. The guanine-nucleotide-releasing protein in the donor membrane recognizes a specific Rab protein and induces it to exchange GDP for GTP. This exchange alters the conforma-tion of the Rab protein, exposing its covalently attached lipid group, which helps anchor the protein in the membrane. The Rab- GTP remains bound to the surface of the transport vesicle after it pinches off from the donor membrane. v-SNARE on the vesicle surface binds to t-SNARE in the target membrane, docking the vesicle. The Rab protein now hydrolyzes its bound GTP, locking the vesicle onto the target membrane and releasing Rab-GDP into the cytosol, from where it can be reused in a new round of transport. The vesicle then fuses with the target membrane. Note that the vesicle coats have been omitted from the drawings for clarity.
Figure 13-58. A current model of protein-mediated vesicle fusion. A complex membrane-fusion machine catalyzes the fusion of a transport vesicle with its target membrane. Only two of the protein components of the fusion complex have been characterized: NSF (N-ethylmaleimide- sensitive fusion protein) and SNAPs (soluble NSF attachment proteins). (NEM is a chemical that modifies free SH groups exposed on protein surfaces and thereby inactivates proteins whose exposed SH groups are required for activity.) SNAREs were first identified as SNAP receptors (hence their name): they bind to both v-SNAREs and t-SNAREs. The binding of the SNAPs allows NSF to bind. This complex, with the help of acyl CoA and as yet unidentified proteins, catalyzes the fusion of the two lipid bilayers. NSF is an ATPase that hydrolyzes ATP to release the complex once it has done its job (not shown).
Figure 13-59. The entry of fowl plague virus into cells. (A) Electron micrographs showing how the virus is endocytosed in a clathrin-coated vesicle, is delivered to an endosome, and then escapes by fusing with the endosomal membrane. (B) Schematic drawing showing how fusion proteins on the surface of the virus mediate its escape from the endosome. (A, Courtesy of Karl Matlin and Hubert Reggio, from K.S. Matlin et al., J. Cell Biol. 91:601-613, 1981, by copyright permission of the Rockefeller University Press.)
Figure 13-60. A model for how a membrane-fusion protein catalyzes lipid bilayer fusion. A cell that expresses the influenza fusion protein on its surface rapidly fuses with neighboring cells after exposure to low pH. The fusion process proceeds through an intermediate (D and E) in which only the outer leaflets of the membranes are fused, while the inner two leaflets are still separate. Indeed, mutant forms of the fusion protein have been obtained that allow the reaction to proceed only to this intermediate state.
14. Energy Conversion: Mitochondria and Chloroplasts
Introduction The Mitochondrion
The Respiratory Chain and ATP Synthase Chloroplasts and Photosynthesis The Evolution of Electron-Transport Chains The Genomes of Mitochondria and Chloroplasts
References