Intracellular Membrane Traffic PDF

695 chapter Intracellular Membrane Traffic Every cell must eat, communicate with the world around it, and quickly respond to changes in its environment. To help accomplish these tasks, cells continually adjust the composition of their plasma membrane and internal compartments in rapid response to...

695 chapter Intracellular Membrane Traffic Every cell must eat, communicate with the world around it, and quickly respond to changes in its environment. To help accomplish these tasks, cells continually adjust the composition of their plasma membrane and internal compartments in rapid response to need. They use an elaborate internal membrane system to add and remove cell-surface proteins, such as receptors, ion channels, and transporters (Figure 13–1). Through the process of exocytosis, the secretory pathway delivers newly synthesized proteins, carbohydrates, and lipids either to the plasma membrane or the extracellular space. By the converse process of endocytosis, cells remove plasma membrane components and deliver them to internal compartments called endosomes, from where they can be recycled to the same or different regions of the plasma membrane or be delivered to lysosomes for degradation. Cells also use endocytosis to capture important nutrients, such as vitamins, cholesterol, and iron; these are taken up together with the macromolecules to which they bind and are then moved on to endosomes and lysosomes, from where they can be transported into the cytosol for use in various biosynthetic processes. The interior space, or lumen, of each membrane-enclosed compartment along the secretory and endocytic pathways is equivalent to the lumen of most other membrane-enclosed compartments and to the cell exterior, in the sense that proteins can travel in this space without having to cross a membrane as they are passed from one compartment to another by means of numerous membrane-enclosed transport containers. These containers are formed from the donor compartment and are either small, spherical vesicles, larger irregular vesicles, or tubules. We shall use the term transport vesicle to apply to all forms of these containers. Within a eukaryotic cell, transport vesicles continually bud off from one membrane and fuse with another, carrying membrane components and soluble lumenal molecules, which are referred to as cargo (Figure 13–2). This vesicular traffic flows along highly organized, directional routes, which allow the cell to secrete, eat, and remodel its plasma membrane and organelles. The secretory pathway leads outward from the endoplasmic reticulum (ER) toward the Golgi apparatus and cell surface, with a side route leading to lysosomes, while the endocytic pathway leads inward from the plasma membrane. In each case, retrieval pathways 13 In This Chapter The Molecular Mechanisms of Membrane Transport and the Maintenance of Compartmental Diversity Transport from the ER Through the Golgi Apparatus Transport from the Trans Golgi Network to Lysosomes Transport into the Cell from the Plasma Membrane: Endocytosis Transport from the Trans Golgi Network to the Cell Exterior: Exocytosis plasma membrane CYTOSOL (A) exocytosis CYTOSOL (B) endocytosis Figure 13–1 Exocytosis and endocytosis. (A) In exocytosis, a transport vesicle fuses with the plasma membrane. Its content is released into the extracellular space, while the vesicle membrane (red) becomes continuous with the plasma membrane. (B) In endocytosis, a plasma membrane patch (red) is internalized, forming a transport vesicle. Its content derives from the extracellular space. Chapter 13: Intracellular Membrane Traffic 696 CYTOSOL DONOR COMPARTMENT LUMEN FUSION cargo molecules BUDDING TARGET COMPARTMENT balance the flow of membrane between compartments in the opposite direction, bringing membrane and selected proteins back to the compartment of origin (Figure 13–3). To perform its function, each transport vesicle that buds from a compartment must be selective. It must take up only the appropriate molecules and must fuse only with the appropriate target membrane. A vesicle carrying cargo from the ER to the Golgi apparatus, for example, must exclude most proteins that are to stay in the ER, and it must fuse only with the Golgi apparatus and not with any other organelle. We begin this chapter by considering the molecular mechanisms of budding 13.02/13.02 and fusion that underlie all vesicle transport.MBOC6 We then discuss the fundamental problem of how, in the face of this transport, the cell maintains the molecular and ENDOPLASMIC RETICULUM lysosome Figure 13–2 Vesicle transport. Transport vesicles bud off from one compartment and fuse with another. As they do so, they carry material as cargo from the lumen (the space within a membrane-enclosed compartment) and membrane of the donor compartment to the lumen and membrane of the target compartment, as shown. EXTRACELLULAR SPACE CYTOSOL CYTOSOL late endosome GOLGI endocytic vesicle plasma membrane nuclear envelope LATE ENDOSOME SECRETORY VESICLES endoplasmic reticulum early endosome LYSOSOME recycling endosome EARLY ENDOSOME RECYCLING ENDOSOME plasma membrane cisternae EXTRACELLULAR SPACE (A) Golgi apparatus secretory vesicle (B) Figure 13–3 A “road-map” of the secretory and endocytic pathways. (A) In this schematic roadmap, which was introduced in Chapter 12, the endocytic and secretory pathways are illustrated with green and red arrows, respectively. In addition, blue arrows denote retrieval pathways for the backflow of selected components. (B) The compartments of the eukaryotic cell involved in vesicle transport. The lumen of each membrane-enclosed compartment is topologically equivalent to the outside of the cell. All compartments shown communicate with one another and the outside of the cell by means of transport vesicles. In the secretory pathway (red arrows), protein molecules are transported from the ER to the plasma membrane or (via endosomes) to lysosomes. In the endocytic pathway (green arrows), molecules are ingested in endocytic vesicles derived from the plasma membrane and delivered to early endosomes and then (via late endosomes) to lysosomes. Many endocytosed molecules are retrieved from early endosomes and returned (some via recycling endosomes) to the cell surface for reuse; similarly, some molecules are retrieved from the early and late endosomes and returned to the Golgi apparatus, and some are retrieved from the Golgi apparatus and returned to the ER. All of these retrieval pathways are shown with blue arrows, as in part (A). MBoC6 m13.03/13.03 THE MOLECULAR MECHANISMS OF MEMBRANE TRANSPORT functional differences between its compartments. Finally, we consider the function of the Golgi apparatus, lysosomes, secretory vesicles, and endosomes, as we trace the pathways that connect these organelles. The Molecular Mechanisms of Membrane Transport and the Maintenance of Compartmental Diversity Vesicle transport mediates a continuous exchange of components between the ten or more chemically distinct, membrane-enclosed compartments that collectively comprise the secretory and endocytic pathways. With this massive exchange, how can each compartment maintain its special identity? To answer this question, we must first consider what defines the character of a compartment. Above all, it is the composition of the enclosing membrane: molecular markers displayed on the cytosolic surface of the membrane serve as guidance cues for incoming traffic to ensure that transport vesicles fuse only with the correct compartment. Many of these membrane markers, however, are found on more than one compartment, and it is the specific combination of marker molecules that gives each compartment its molecular address. How are these membrane markers kept at high concentration on one compartment and at low concentration on another? To answer this question, we need to consider how patches of membrane, enriched or depleted in specific membrane components, bud off from one compartment and transfer to another. We begin by discussing how cells segregate proteins into separate membrane domains by assembling a special protein coat on the membrane’s cytosolic face. We consider how coats form, what they are made of, and how they are used to extract specific cargo components from a membrane and compartment lumen for delivery to another compartment. Finally, we discuss how transport vesicles dock at the appropriate target membrane and then fuse with it to deliver their cargo. There Are Various Types of Coated Vesicles Most transport vesicles form from specialized, coated regions of membranes. They bud off as coated vesicles, which have a distinctive cage of proteins covering their cytosolic surface. Before the vesicles fuse with a target membrane, they discard their coat, as is required for the two cytosolic membrane surfaces to interact directly and fuse. The coat performs two main functions that are reflected in a common two-layered structure. First, an inner coat layer concentrates specific membrane proteins in a specialized patch, which then gives rise to the vesicle membrane. In this way, the inner layer selects the appropriate membrane molecules for transport. Second, an outer coat layer assembles into a curved, basketlike lattice that deforms the membrane patch and thereby shapes the vesicle. There are three well-characterized types of coated vesicles, distinguished by their major coat proteins: clathrin-coated, COPI-coated, and COPII-coated (Figure 13–4). Each type is used for different transport steps. Clathrin-coated vesicles, for example, mediate transport from the Golgi apparatus and from the plasma membrane, whereas COPI- and COPII-coated vesicles most commonly mediate transport from the ER and from the Golgi cisternae (Figure 13–5). There is, however, much more variety in coated vesicles and their functions than this short list suggests. As we discuss below, there are several types of clathrin-coated vesicles, each specialized for a different transport step, and the COPI- and COPII-coated vesicles may be similarly diverse. The Assembly of a Clathrin Coat Drives Vesicle Formation Clathrin-coated vesicles, the first coated vesicles to be identified, transport material from the plasma membrane and between endosomal and Golgi compartments. COPI-coated vesicles and COPII-coated vesicles transport material early 697 Chapter 13: Intracellular Membrane Traffic 698 Figure 13–4 Electron micrographs of clathrin-coated, COPI-coated, and COPII-coated vesicles. All are shown in electron micrographs at the same scale. (A Clathrin-coated vesicles. (B) COPIcoated vesicles and Golgi cisternae (red arrows) from a cell-free system in which COPI-coated vesicles bud in the test tube. (C) COPII-coated vesicles. (A and B, from L. Orci, B. Glick and J. Rothman, Cell 46:171–184, 1986. With permission from Elsevier; C, courtesy of Charles Barlowe and Lelio Orci.) (A) clathrin (B) COPI (C) COPII 100 nm in the secretory pathway: COPI-coated vesicles bud from Golgi compartments, and COPII-coated vesicles bud from the ER (see Figure 13–5). We discuss clathrin-coated vesicles first, as they provide a good example of how vesicles form. The major protein component of clathrin-coated vesicles is clathrin itself, which forms the outer layer of the coat. Each clathrin subunit consists of three large and three small polypeptide chains that together form a three-legged structure called a triskelion (Figure 13–6A,B). Clathrin triskelions assemble into a basketlike framework of hexagons and pentagons to form coated pits (buds) on the m13.04/13.04 cytosolic surface of membranesMBoC6 (Figure 13–7). Under appropriate conditions, isolated triskelions spontaneously self-assemble into typical polyhedral cages in a test tube, even in the absence of the membrane vesicles that these baskets normally enclose (Figure 13–6C,D). Thus, the clathrin triskelions determine the geometry of the clathrin cage (Figure 13–6E). Adaptor Proteins Select Cargo into Clathrin-Coated Vesicles Adaptor proteins, another major coat component in clathrin-coated vesicles, form a discrete inner layer of the coat, positioned between the clathrin cage and the membrane. They bind the clathrin coat to the membrane and trap various transmembrane proteins, including transmembrane receptors that capture soluble cargo molecules inside the vesicle—so-called cargo receptors. In this way, the adaptor proteins select a specific set of transmembrane proteins, together with the soluble proteins that interact with them, and package them into each newly formed clathrin-coated transport vesicle (Figure 13–8). late endosome KEY: early endosome clathrin EXTRACELLULAR SPACE COPI COPII CYTOSOL trans Golgi Golgi cisternae network ER Golgi apparatus secretory vesicle plasma membrane Figure 13–5 Use of different coats for different steps in vesicle traffic. Different coat proteins select different cargo and shape the transport vesicles that mediate the various steps in the secretory and endocytic pathways. When the same coats function in different places in the cell, they usually incorporate different coat protein subunits that modify their properties (not shown). Many differentiated cells have additional pathways beside those shown here, including a sorting pathway from the trans Golgi network to the apical surface of epithelial cells and a specialized recycling pathway for proteins of synaptic vesicles in the nerve terminals of neurons (see Figure 11–36). The arrows are colored as in Figure 13–3. THE MOLECULAR MECHANISMS OF MEMBRANE TRANSPORT 699 (A) light chain heavy chains (B) (C) (D) (E) 50 nm 25 nm Figure 13–6 The structure of a clathrin coat. (A) Electron micrograph of a clathrin triskelion shadowed with platinum. (B) Each triskelion is composed of three clathrin heavy chains and three clathrin light chains, as shown in the diagram. (C and D) A cryoelectron micrograph taken of a clathrin coat composed of 36 triskelions organized in a network of 12 pentagons and 6 hexagons, with some heavy chains (C) and light chains (D) highlighted (Movie 13.1). The light chains link to the actin cytoskeleton, which helps generate force for membrane budding and vesicle movement, and their phosphorylation regulates clathrin coat assembly. The interwoven legs of the clathrin triskelions form an outer shell from which the N-terminal domains of the triskelions protrude inward. These domains bind to the adaptor proteins shown in Figure 13–8. The coat shown was assembled biochemically from pure clathrin triskelions and is too small to enclose a membrane vesicle. (E) Images of clathrincoated vesicles isolated from bovine brain. The clathrin coats are constructed in a similar but less regular way, from pentagons, a larger number of hexagons, and sometime resembling the architecture of deformed soccer balls. The structures MBoC6 heptagons, m13.07/13.06 were determined by cryoelectron microscopy and tomographic reconstruction. (A, from E. Ungewickell and D. Branton, Nature 289:420–422, 1981; C and D, from A. Fotin et al., Nature 432:573–579, 2004. All with permission from Macmillan Publishers Ltd; E, from Y. Cheng et al., J. Mol. Biol. 365:892–899, 2007. With permission from Elsevier.) There are several types of adaptor proteins. The best characterized have four different protein subunits; others are single-chain proteins. Each type of adaptor protein is specific for a different set of cargo receptors. Clathrin-coated vesicles budding from different membranes use different adaptor proteins and thus package different receptors and cargo molecules. The assembly of adaptor proteins on the membrane is tightly controlled, in part by the cooperative interaction of the adaptor proteins with other components of the coat. The adaptor protein AP2 serves as a well-understood example. When it binds to a specific phosphorylated phosphatidylinositol lipid (a phosphoinositide), it alters its conformation, exposing binding sites for cargo receptors in the membrane. The simultaneous binding to the cargo receptors and lipid head groups greatly enhances the binding of AP2 to the membrane (Figure 13–9). Because several requirements must be met simultaneously to stably bind AP2 proteins to a membrane, the proteins act as coincidence detectors that only assemble at the right time and place. Upon binding, they induce membrane curvature, which makes the binding of additional AP2 proteins in its proximity more likely. The cooperative assembly of the AP2 coat layer then is further amplified by clathrin binding, which leads to the formation and budding of a transport vesicle. Adaptor proteins found in other coats also bind to phosphoinositides, which not only have a major role in directing when and where coats assemble in the cell, but also are used much more widely as molecular markers of compartment identity. This helps to control vesicular traffic, as we now discuss. Figure 13–7 Clathrin-coated pits and vesicles. This rapid-freeze, deepetch 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 expose the cytoplasmic surface of the plasma membrane. (Courtesy of John Heuser.) 0.2 µm 700 Chapter 13: Intracellular Membrane Traffic coated vesicle membrane clathrin triskelion donor membrane cargo receptor adaptor protein adaptor protein naked transport vesicle CYTOSOL cargo molecules COAT ASSEMBLY AND CARGO SELECTION membrane-bending and fission proteins BUD FORMATION VESICLE FORMATION Phosphoinositides Mark Organelles and Membrane Domains Although inositol phospholipids typically comprise less than 10% of the total phospholipids in a membrane, they have important regulatory functions. They can undergo rapid cycles of phosphorylation and dephosphorylation at the 3ʹ, 4ʹ, and 5ʹ positions of their inositol sugar head groups to produce various types of phosphoinositides (phosphatidylinositol phosphates, or PIPs). The interconversion of phosphatidylinositol (PI) and PIPs is highly compartmentalized: different organelles in the endocytic and secretory pathways have distinct sets of PI and PIP kinases and PIP phosphatases (Figure 13–10). The distribution, regulation, and local balance of these enzymes determine the steady-state distribution of each PIP species. As a consequence, the distribution of PIPs varies from organelle to organelle, and often within a continuous membrane from one region to another, thereby defining specialized membrane domains. Many proteins involved at different steps in vesicle contain domains MBoC6transport m13.08/13.08 that bind with high specificity to the head groups of particular PIPs, distinguishing one phosphorylated form from another (see Figure 13–10 E and F). Local control of the PI and PIP kinases and PIP phosphatases can therefore be used to rapidly control the binding of proteins to a membrane or membrane domain. The production of a particular type of PIP recruits proteins containing matching PIP-binding domains. The PIP-binding proteins then help regulate vesicle formation and other steps in the control of vesicle traffic (Figure 13–11). The same strategy is widely used to recruit specific intracellular signaling proteins to the plasma membrane in response to extracellular signals (discussed in Chapter 15). cargo receptors phosphoinositide PI(4,5)P2 endocytosis signals β2 µ2 σ2 α AP2 locked AP2 open UNCOATING Figure 13–8 The assembly and disassembly of a clathrin coat. The assembly of the coat introduces curvature into the membrane, which leads in turn to the formation of a coated bud (called a coated pit if it is in the plasma membrane). The adaptor proteins bind both clathrin triskelions and membrane-bound cargo receptors, thereby mediating the selective recruitment of both membrane and soluble cargo molecules into the vesicle. Other membrane-bending and fission proteins are recruited to the neck of the budding vesicle, where sharp membrane curvature is introduced. The coat is rapidly lost shortly after the vesicle buds off. Figure 13–9 Lipid-induced conformation switching of AP2. The AP2 adaptor protein complex has four subunits (α, β2, μ2, and σ2). Upon interaction with the phosphoinositide PI(4,5)P2 (see Figure 13–10) in the cytosolic leaflet of the plasma membrane, AP2 rearranges so that binding sites for cargo receptors become exposed. Each AP2 complex binds four PI(4,5)P2 molecules (for clarity, only one is shown). In the open AP2 complex, the μ2 and σ2 subunits bind the cytosolic tails of cargo receptors that display the appropriate endocytosis signals. These signals consist of short amino acid sequence motifs. When AP2 binds tightly to the membrane, it induces curvature, which favors the binding of additional AP2 complexes in the vicinity. THE MOLECULAR MECHANISMS OF MEMBRANE TRANSPORT HO OH HO 4 HO OH 3 2 5 6 O P O CH2 CH CH2 O PI(3)P PI PI(5)P PI(3,4)P2 PI(4)P PI(4,5)P2 _ O O PI(3,5)P2 1 P O 701 PI (B) O C O C (D) PI(3,4,5)P3 P P 4 3 2 5 6 1 P PI(3,4)P2 (A) (C) (E) (F) Membrane-Bending Proteins Help Deform the Membrane During Vesicle Formation The forces generated by clathrin coat assembly alone are not sufficient to shape and pinch off a vesicle from the membrane. Other membrane-bending MBoC6 m13.10/13.09 and force-generating proteins participate at every stage of the process. Membrane-bending proteins that contain crescent-shaped domains, called BAR domains, bind to and impose their shape on the underlying membrane via electrostatic interactions with the lipid head groups (Figure 13–12; also see Figure 10–40). Such BAR-domain proteins are thought to help AP2 nucleate clathrin-mediated endocytosis by shaping the plasma membrane to allow a clathrin-coated bud to form. Some of these proteins also contain amphiphilic helices that induce membrane curvature after being inserted as wedges into the cytoplasmic leaflet of the membrane. Other BAR-domain proteins are important in shaping the neck of a budding vesicle, where stabilization of sharp membrane bends is essential. Finally, the clathrin machinery nucleates the local assembly of actin filaments that introduce tension to help pinch off and propel the forming vesicle away from the membrane. Figure 13–10 Phosphatidylinositol (PI) and phosphoinositides (PIPs). (A, B) The structure of PI shows the free hydroxyl groups in the inositol sugar that can in principle be modified. (C) Phosphorylation of one, two, or three of the hydroxyl groups on PI by PI and PIP kinases produces a variety of PIP species. They are named according to the ring position (in parentheses) and the number of phosphate groups (subscript) added to PI. PI(3,4)P2 is shown. (D) Animal cells have several PI and PIP kinases and a similar number of PIP phosphatases, which are localized to different organelles, where they are regulated to catalyze the production of particular PIPs. The red and green arrows show the kinase and phosphatase reactions, respectively. (E, F) Phosphoinositide head groups are recognized by protein domains that discriminate between the different forms. In this way, select groups of proteins containing such domains are recruited to regions of membrane in which these phosphoinositides are present. PI(3)P and PI(4,5)P2 are shown. (D, modified from M.A. de Matteis and A. Godi, Nat. Cell Biol. 6:487–492, 2004. With permission from Macmillan Publishers Ltd.) Cytoplasmic Proteins Regulate the Pinching-Off and Uncoating of Coated Vesicles As a clathrin-coated bud grows, soluble cytoplasmic proteins, including dynamin, assemble at the neck of each bud (Figure 13–13). Dynamin contains a PI(4,5) P2-binding domain, which tethers the protein to the membrane, and a GTPase domain, which regulates the rate at which vesicles pinch off from the membrane. Figure 13–11 The intracellular location of phosphoinositides. Different types of PIPs are located in different membranes and membrane domains, where they are often associated with specific vesicle transport events. The membrane of secretory vesicles, for example, contains PI(4)P. When the vesicles fuse with the plasma membrane, a PI 5-kinase that is localized there converts the PI(4)P into PI(4,5)P2. The PI(4,5)P2, in turn, helps recruit adaptor proteins, which initiate the formation of a clathrin-coated pit, as the first step in clathrin-mediated endocytosis. Once the clathrin-coated vesicle buds off from the plasma membrane, a PI(5)P phosphatase hydrolyzes PI(4,5)P2, which weakens the binding of the adaptor proteins, promoting vesicle uncoating. We discuss phagocytosis and the distinction between regulated and constitutive exocytosis later in the chapter. (Modified from M.A. de Matteis and A. Godi, Nat. Cell Biol. 6:487–492, 2004. With permission from Macmillan Publishers Ltd.) phagocytosis endocytosis regulated exocytosis constitutive exocytosis KEY: PI(3)P PI(4)P PI(4,5)P2 PI(3,5)P2 PI(3,4,5)P3 702 Chapter 13: Intracellular Membrane Traffic BAR domain dimer + membrane The pinching-off process brings the two noncytosolic leaflets of the membrane into close proximity and fuses them, sealing off the forming vesicle (see Figure 13–2). To perform this task, dynamin recruits other proteins to the neck of the bud. Together with dynamin, they help bend the patch of membrane—by directly MBoC6 n13.100/13.11 distorting the bilayer structure, or by changing its lipid composition through the recruitment of lipid-modifying enzymes, or by both mechanisms. Once released from the membrane, the vesicle rapidly loses its clathrin coat. A PIP phosphatase that is co-packaged into clathrin-coated vesicles depletes PI(4,5) P2 from the membrane, which weakens the binding of the adaptor proteins. In addition, an hsp70 chaperone protein (see Figure 6–80) functions as an uncoating ATPase, using the energy of ATP hydrolysis to peel off the clathrin coat. Auxilin, another vesicle protein, is thought to activate the ATPase. The release of the coat, however, must not happen prematurely, so additional control mechanisms must somehow prevent the clathrin from being removed before it has formed a complete vesicle (discussed below). Figure 13–12 The structure of BAR domains. BAR-domain proteins are diverse and enable many membrane-bending processes in the cell. BAR domains are built from coiled coils that dimerize into modules with a positively charged inner surface, which preferentially interacts with negatively charged lipid head groups to bend membranes. Local membrane deformations caused by BAR-domain proteins facilitate the binding of additional BAR-domain proteins, thereby generating a positive feedback cycle for curvature propagation. Individual BAR-domain proteins contain a distinctive curvature and often have additional features that adapt them to their specific tasks: some have short amphiphilic helices that cause further membrane deformation by wedge insertion; others are flanked by PIP-binding domains that direct them to membranes enriched in cognate phosphoinositides. constricted membrane neck GTPase domain (C) (A) dynamin helix and associated proteins GTP HYDROLYSIS GTPase domain of dynamin (D) (B) 200 nm Figure 13–13 The role of dynamin in pinching off clathrin-coated vesicles. (A) Multiple dynamin molecules assemble into a spiral around the neck of the forming bud. The dynamin spiral is thought to recruit other proteins to the bud neck, which, together with dynamin, destabilize the interacting lipid bilayers so that the noncytoplasmic leaflets flow together. The newly formed vesicle then pinches off from the membrane. Specific mutations in dynamin can either enhance or block the pinching-off process. (B) Dynamin was discovered as the protein defective in the shibire mutant of Drosophila. These mutant flies become paralyzed because clathrin-mediated endocytosis stops, and the synaptic vesicle membrane fails to recycle, blocking neurotransmitter release. Deeply invaginated clathrin-coated pits form in the nerve endings of the fly’s nerve cells, with a belt of mutant dynamin assembled around the neck, as shown in this thin-section electron micrograph. The pinching-off process fails because the required MBoC6 m13.12/13.12 membrane fusion does not take place. (C, D) A model of how conformational changes in the GTPase domains of membrane-assembled dynamin may power a conformational change that constricts the neck of the bud. A single dynamin molecule is shown in orange in D. (B, from J.H. Koenig and K. Ikeda, J. Neurosci. 9:3844–3860, 1989. With permission from the Society of Neuroscience; C and D, adapted from M.G.J. Ford, S. Jenni and J. Nunnari, Nature 477:561–566, 2011. With permission from Macmillan Publishers.) THE MOLECULAR MECHANISMS OF MEMBRANE TRANSPORT Monomeric GTPases Control Coat Assembly To balance the vesicle traffic to and from a compartment, coat proteins must assemble only when and where they are needed. While local production of PIPs plays a major part in regulating the assembly of clathrin coats on the plasma membrane and Golgi apparatus, cells superimpose additional ways of regulating coat formation. Coat-recruitment GTPases, for example, control the assembly of clathrin coats on endosomes and the COPI and COPII coats on Golgi and ER membranes. Many steps in vesicle transport depend on a variety of GTP-binding proteins that control both the spatial and temporal aspects of vesicle formation and fusion. As discussed in Chapter 3, GTP-binding proteins regulate most processes in eukaryotic cells. They act as molecular switches, which flip between an active state with GTP bound and an inactive state with GDP bound. Two classes of proteins regulate the flipping: guanine nucleotide exchange factors (GEFs) activate the proteins by catalyzing the exchange of GDP for GTP, and GTPase-activating proteins (GAPs) inactivate the proteins by triggering the hydrolysis of the bound GTP to GDP (see Figures 3–68 and 15–7). Although both monomeric GTP-binding proteins (monomeric GTPases) and trimeric GTP-binding proteins (G proteins) have important roles in vesicle transport, the roles of the monomeric GTPases are better understood, and we focus on them here. Coat-recruitment GTPases are members of a family of monomeric GTPases. They include the ARF proteins, which are responsible for the assembly of both COPI and clathrin coats assembly at Golgi membranes, and the Sar1 protein, which is responsible for the assembly of COPII coats at the ER membrane. Coat-recruitment GTPases are usually found in high concentration in the cytosol in an inactive, GDP-bound state. When a COPII-coated vesicle is to bud from the ER membrane, for example, a specific Sar1-GEF embedded in the ER membrane binds to cytosolic Sar1, causing the Sar1 to release its GDP and bind GTP in its place. (Recall that GTP is present in much higher concentration in the cytosol than GDP and therefore will spontaneously bind after GDP is released.) In its GTP-bound state, the Sar1 protein exposes an amphiphilic helix, which inserts into the cytoplasmic leaflet of the lipid bilayer of the ER membrane. The tightly bound Sar1 now recruits adaptor coat protein subunits to the ER membrane to initiate budding (Figure 13–14). Other GEFs and coat-recruitment GTPases operate in a similar way on other membranes. The coat-recruitment GTPases also have a role in coat disassembly. The hydrolysis of bound GTP to GDP causes the GTPase to change its conformation so that its hydrophobic tail pops out of the membrane, causing the vesicle’s coat to disassemble. Although it is not known what triggers the GTP hydrolysis, it has been proposed that the GTPases work like timers, which hydrolyze GTP at slow but predictable rates, to ensure that vesicle formation is synchronized with the requirements of the moment. COPII coats accelerate GTP hydrolysis by Sar1, and a fully formed vesicle will be produced only when bud formation occurs faster than the timed disassembly process; otherwise, disassembly will be triggered before a vesicle pinches off, and the process will have to start again, perhaps at a more appropriate time and place. Once a vesicle pinches off, GTP hydrolysis releases Sar1, but the sealed coat is sufficiently stabilized through many cooperative interactions, including binding to the cargo receptors in the membrane, that it may stay on the vesicle until the vesicle docks at a target membrane. There, a kinase phosphorylates the coat proteins, which completes coat disassembly and readies the vesicle for fusion. Clathrin- and COPI-coated vesicles, by contrast, shed their coat soon after they pinch off. For COPI vesicles, the curvature of the vesicle membrane serves as a trigger to begin uncoating. An ARF-GAP is recruited to the COPI coat as it assembles. It interacts with the membrane, and senses the lipid packing density. It becomes activated when the curvature of the membrane approaches that of a transport vesicle. It then inactivates ARF, causing the coat to disassemble. 703 Chapter 13: Intracellular Membrane Traffic 704 inactive, soluble Sar1-GDP GDP GDP amphiphilic helix GTP active, membranebound Sar1-GTP GTP Sec24 Sec23 ER membrane GTP Sar1-GTP CYTOSOL (A) cargo receptor donor membrane (ER) ER LUMEN Sar1-GEF (B) cargo COPII-coated vesicle Sec13/31 Sec23/24 Sar1-GTP (C) 25 nm (D) selected membrane proteins outer coat inner coat donor membrane Not All Transport Vesicles Are Spherical Although vesicle-budding is similar at various locations in the cell, each cell membrane poses its own special challenges. The plasma membrane, for example, is comparatively flat and stiff, owing to its cholesterol-rich lipid composition and underlying actin-rich cortex. Thus, the coordinated action of clathrin coats and membrane-bending proteins has to produce sufficient force to introduce curvature, especially at the neck of the bud where sharp bends are required for the pinching-off processes. In contrast, vesicle-budding from many intracellular membranes occurs preferentially at regions where the membranes are already curved, such as the rimsMBoC6 of the m13.13/13.13 Golgi cisternae or ends of membrane tubules. In these places, the primary function of the coats is to capture the appropriate cargo proteins rather than to deform the membrane. Transport vesicles also occur in various sizes and shapes. Diverse COPII vesicles are required for the transport of large cargo molecules. Collagen, for example, is assembled in the ER as 300-nm-long, stiff procollagen rods that then are secreted from the cell where they are cleaved by proteases to collagen, which is embedded into the extracellular matrix (discussed in Chapter 19). Procollagen rods do not fit into the 60–80 nm COPII vesicles normally observed. To circumvent this problem, the procollagen cargo molecules bind to transmembrane packaging proteins in the ER, which control the assembly of the COPII coat components (Figure 13–15). These events drive the local assembly of much larger COPII vesicles that accommodate the oversized cargo. Human mutations in genes encoding such packaging proteins result in collagen defects with severe consequences, such as skeletal abnormalities and other developmental defects. Similar mechanisms must regulate the sizes of vesicles required to secrete other large macromolecular complexes, including the lipoprotein particles that transport lipids out of cells. Figure 13–14 Formation of a COPIIcoated vesicle. (A) Inactive, soluble Sar1-GDP binds to a Sar1-GEF in the ER membrane, causing the Sar1 to release its GDP and bind GTP. A GTP-triggered conformational change in Sar1 exposes an amphiphilic helix, which inserts into the cytoplasmic leaflet of the ER membrane, initiating membrane bending (which is not shown). (B) GTP-bound Sar1 binds to a complex of two COPII adaptor coat proteins, called Sec23 and Sec24, which form the inner coat. Sec24 has several different binding sites for the cytosolic tails of cargo receptors. The entire surface of the complex that attaches to the membrane is gently curved, matching the diameter of COPII-coated vesicles. (C) A complex of two additional COPII coat proteins, called Sec13 and Sec 31, forms the outer shell of the coat. Like clathrin, they can assemble on their own into symmetrical cages with appropriate dimensions to enclose a COPII-coated vesicle. (D) Membrane-bound, active Sar1-GTP recruits COPII adaptor proteins to the membrane. They select certain transmembrane proteins and cause the membrane to deform. The adaptor proteins then recruit the outer coat proteins which help form a bud. A subsequent membrane fusion event pinches off the coated vesicle. Other coated vesicles are thought to form in a similar way. (C, modified from S.M. Stagg et al., Nature 439:234–238, 2006. With permission from Macmillan Publishers Ltd.) THE MOLECULAR MECHANISMS OF MEMBRANE TRANSPORT 705 Sec13/31 CYTOSOL packaging proteins Sec23 Sec24 ER LUMEN procollagen Many other vesicle budding events likewise involve variations of common mechanisms. When living cells are genetically engineered to express fluorescent membrane components, the endosomes and trans Golgi network are seen MBoC6 n13.105/13.14 in a fluorescence microscope to continually send out long tubules. Coat proteins assemble onto the membrane tubules and help recruit specific cargo. The tubules then either regress or pinch off with the help of dynamin-like proteins to form transport vesicles of different sizes and shapes. Tubules have a higher surface-to-volume ratio than the larger organelles from which they form. They are therefore relatively enriched in membrane proteins compared with soluble cargo proteins. As we discuss later, this property of tubules is an important feature for sorting proteins in endosomes. Rab Proteins Guide Transport Vesicles to Their Target Membrane To ensure an orderly flow of vesicle traffic, transport vesicles must be highly accurate in recognizing the correct target membrane with which to fuse. Because of the diversity and crowding of membrane systems in the cytoplasm, a vesicle is likely to encounter many potential target membranes before it finds the correct one. Specificity in targeting is ensured because all transport vesicles display surface markers that identify them according to their origin and type of cargo, and target membranes display complementary receptors that recognize the appropriate markers. This crucial process occurs in two steps. First, Rab proteins and Rab effectors direct the vesicle to specific spots on the correct target membrane. Second, SNARE proteins and SNARE regulators mediate the fusion of the lipid bilayers. Rab proteins play a central part in the specificity of vesicle transport. Like the coat-recruitment GTPases discussed earlier (see Figure 13–14), Rab proteins are also monomeric GTPases. With over 60 known members, the Rab subfamily is the largest of the monomeric GTPase subfamilies. Each Rab protein is associated with one or more membrane-enclosed organelles of the secretory or endocytic pathways, and each of these organelles has at least one Rab protein on its cytosolic surface (Table 13–1). Their highly selective distribution on these membrane systems makes Rab proteins ideal molecular markers for identifying each membrane type and guiding vesicle traffic between them. Rab proteins can function on transport vesicles, on target membranes, or both. Like the coat-recruitment GTPases, Rab proteins cycle between a membrane and the cytosol and regulate the reversible assembly of protein complexes on the membrane. In their GDP-bound state, they are inactive and bound to another protein (Rab-GDP dissociation inhibitor, or GDI) that keeps them soluble in the Figure 13–15 Packaging of procollagen into large tubular COPII-coated vesicles. The cartoons show models for two COPII coat assembly modes. The models are based on cryoelectron tomography images of reconstituted COPII vesicles. On a spherical membrane (left), the Sec23/24 inner coat proteins assemble in patches that anchor the Sec13/31 outer coat protein cage. The Sec13/31 rods assemble a cage of triangles, squares, and pentagons. When procollagen needs to be packaged (right), special packaging proteins sense the cargo and modify the coat assembly process. This interaction recruits the COPII inner coat protein Sec24 and locally enhances the rate with which Sar1 cycles on and off the membrane (not shown). In addition, a monoubiquitin is added to the Sec31 protein, changing the assembly properties of the outer cage. Sec23/24 proteins arrange in larger arrays and Sec13/31 arrange in a regular lattice of diamond shapes. As the result, a large tubular vesicle is formed that can accommodate the large cargo molecules. The packaging proteins are not part of the budding vesicle but remain in the ER. (Modified from G. Zanetti et al., eLife 2:e00951, 2013.)