Secretory Membrane System and Golgi Apparatus

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CHAPTER 21 Secretory Membrane System and Golgi Apparatus

Eukaryotic cells transport newly synthesized proteins destined for the extracellular space, the plasma membrane, or the endocytic/lysosomal system through a series of functionally distinct, membrane-bound compartments, including the endoplasmic reticulum (ER), Golgi apparatus, and vesicular transport intermediates. This is the secretory membrane system (Fig. 21-1), which allows eukaryotic cells to perform three major functions: (1) distribute proteins and lipids synthesized in the ER to the cell surface and other cellular sites, (2) modify and/or store protein and lipid molecules after their export from the ER, and (3) generate and maintain the unique identity and function of the ER, Golgi apparatus, and plasma membrane. This chapter describes how the secretory membrane system is organized and operates to fulfill these functions. It also provides a detailed description of the Golgi apparatus whose conserved features are central for the operation of the secretory membrane system.

Figure 21-1 overview of the secretory membrane system. The three principal organelles of the secretory pathway—the ER, Golgi apparatus, and plasma membrane—communicate with one another and the cell exterior by way of transport carriers. The carriers (either small vesicles or larger vesicle-tubule elements) move along cytoskeletal elements (red lines) to transfer newly synthesized proteins, called cargo, from the ER to the Golgi, and from the Golgi to the plasma membrane (or to the endosome/lysosomal system). Carriers form from the ER at specialized regions, called ER export domains, producing pre-Golgi structures called vesicular tubular carriers (VTCs) that move to the Golgi. Retrograde transport carriers bud off from the VTC or Golgi apparatus to retrieve proteins and lipids back to the ER for repeated use and to balance the anterograde flow of membrane to the plasma membrane. The lumenal spaces enclosed by the carriers and organelles of the secretory membrane system are all topologically equivalent to the outside of the cell.

Overview of the Secretory Membrane System

The secretory membrane system uses membrane-enclosed transport carriers to move thousands of diverse macromolecules—including proteins, proteoglycans, and glycoproteins—efficiently and precisely among different membrane-bound compartments (i.e., the ER, Golgi apparatus, and plasma membrane). Within the large cytoplasmic volume of the eukaryotic cell (up to 10³ times that of the volume of a prokaryotic cell), this is essential for coordinating cellular needs in response to the constantly changing environment and organismal physiology.

Newly synthesized transmembrane and lumenal proteins transported through the secretory system are called cargo. These include lumenal proteins destined to be stored within a compartment or secreted to the cell exterior, as well as transmembrane proteins that are retained in a particular compartment (e.g., Golgi processing enzymes), delivered to the plasma membrane, or recycled among compartments (e.g., transport machinery). Transfer of cargo molecules through the secretory system begins with their cotranslational insertion into or across the ER bilayer (see Fig. 20-7). The cargo molecules are next folded and assembled into forms that can be sorted and concentrated within membrane-bound transport intermediates (called vesicular tubular carriers [VTCs]) destined for the Golgi apparatus. Once packaged into and transported by such a carrier, cargo enters the Golgi apparatus, which serves as the central processing and sorting station in the secretory membrane system. Within the Golgi apparatus, numerous enzymes modify the cargo molecules by trimming or elongating the cargo’s glycan side chains or cleaving its polypeptides. Processed cargo is then sorted into membrane-bound carriers that bud out from the Golgi apparatus and move to the plasma membrane, to the endosome/lysosomal system, or back to the ER. In specialized cell types, the Golgi apparatus can sort certain classes of cargo into secretory granules (for storage and later release to the cell exterior in response to specific stimuli) or give rise to transport carriers that target to different polarized plasma membrane domains.

Membrane-enclosed carriers mediate transport within the secretory membrane system (Fig. 21-2). Carriers are shaped as tubules, vesicles, or larger structures. The carriers are too large to diffuse freely in the crowded cytoplasm but are transported over long distances along microtubules or actin filaments by molecular motor proteins. Each carrier selects certain types of cargo before budding from a donor compartment and fuses only with an appropriate target membrane. Molecular markers on the cytoplasmic surface of the carrier, as well as on the acceptor membrane, steer the carrier through the cytoplasm and ensure that it fuses only with the correct target compartment. The carriers continuously shuttle among ER, Golgi apparatus, and plasma membranes, enabling cargo to be distributed to its appropriate target organelle.

Figure 21-2 carrier transport. Membrane-enclosed carriers (shaped as vesicles, tubules, or vesicle-tubule elements) bud off from a donor compartment after packaging both lumenal and transmembrane cargo proteins. Carriers are moved through the cytoplasm along cytoskeletal elements (e.g., microtubules in mammalian cells) by motor proteins until they fuse with a target compartment. During this process, the relative topology of the lipids and transmembrane proteins is maintained.

Sorting of cargo into transport carriers is facilitated by the presence of specialized lipids in the donor organelle membrane (such as sphingomyelin, glycosphingolipids, and phosphoinositides in the Golgi apparatus) and by the recruitment of protein-based sorting and transport machinery (e.g., coat proteins and tethering/fusion factors). Together, the specialized lipids and protein-sorting machinery generate membrane microdomains that concentrate or exclude cargo. The domains then pinch off the membrane bilayer as membrane-enclosed carriers and travel to target membranes.

During transport of a carrier, the relative orientation (called topology) of lipid and protein in the membrane bilayer, established during synthesis in the ER, is maintained (Fig. 21-2). Hence, one side of the membrane always faces the cytoplasm. The other side initially faces the lumen of the ER. This side remains inside each membrane compartment along the secretory pathway but is exposed on the cell surface if the carrier fuses with the plasma membrane. Selection of proteins and lipids by a carrier, budding of the carrier, and subsequent fusion of the carrier with an acceptor compartment all also occur without leakage of contents from the carrier or the donor and target compartments.

The flow of cargo and lipid forward through the secretory system toward the plasma membrane (anterograde traffic) is balanced by selective retrograde traffic of cargo and lipids back toward the ER (Fig. 21-1). Retrograde traffic allows proteins and lipids involved in membrane transport and fusion to be retrieved for repeated use. Retrograde traffic also returns proteins that have been inadvertently carried forward through the secretory system so they can be redirected to their proper destination. Both anterograde and retrograde flows of membrane within the secretory system are necessary for the ER, Golgi apparatus, and plasma membrane to generate and maintain their distinct functional and morphologic identities.

Advantages of the Secretory Membrane System

The secretory membrane system, found in all eukaryotic cells, offers numerous advantages over the simpler secretory process in prokaryotic cells, which involves insertion of newly synthesized proteins directly into or across the plasma membrane. First, synthesizing, folding, and processing membrane and secretory proteins within a series of distinct compartments provides a protective environment for cells to modify proteins before they are exposed on the cell surface. Newly synthesized pro-teins within the ER, for example, can fold into complex shapes and assemble into multisubunit complexes. Within the Golgi apparatus, the cargo molecules can be further modified by glycan processing and proteolytic cleavage. The resulting repertoire of protein structures that are expressed at the cell surface is significantly larger and capable of performing more diverse functions than that found in prokaryotes.

A second advantage is the capacity of the secretory membrane system to regulate protein secretion and expression at the cell surface. Eukaryotic cells can store proteins in membrane compartments before releasing them at the cell surface in response to internal or external signals. By exploiting these capabilities, eukaryotic cells have evolved elaborate ways to control the types of proteins located on or secreted from the cell surface.

A third advantage relates to the differentiation of the plasma membrane. Prokaryotic cells synthesize their proteins at the plasma membrane, so they must keep this surface enriched in loosely packed glycerophospholipids that are pliable enough that newly synthesized proteins can enter into and fold in a hydrophobic environment. Consequently, prokaryotic cells secrete a rigid cell wall as a protective barrier to the outside. In eukaryotes, concentrating protein synthesis in the ER frees the plasma membrane to become enriched in lipids such as cholesterol and sphingolipids that can arrange into highly ordered, flexible arrays. The ordered, flexible arrays of cholesterol and sphingolipids in the plasma membrane provide mechanical stability and an impermeable barrier to water-soluble molecules. As a consequence, eukaryotic cells do not require a cell wall to survive (although some eukaryotes, such as plant and fungal cells, make cell walls) and can employ their plasma membrane in a wide range of functions, such as membrane protrusion for engulfing large extracellular objects (see Chapter 22) and for crawling (see Chapter 38).

Building and Maintaining the Secretory Membrane System

Effective operation of the secretory membrane system depends on several features. The system must generate and maintain the specialized character of each secretory compartment (including the different lipid and protein environments of the ER, Golgi apparatus, and plasma membrane) in the face of continual exchange of protein and lipid components. Cargo must be concentrated selectively in or excluded from each transport carrier. Each carrier must be directed along a specific route and fuse only with an appropriate target membrane.

Two mechanisms, described in more detail in the following sections, play important roles in accomplishing these tasks. First, a lipid-based sorting mechanism uses the inherent capacity of lipids to self-organize into different domains to create a gradient of phospholipid composition across the secretory pathway. On the basis of the length of their transmembrane segments, transmembrane proteins partition into particular membranes that differ in the thickness of the lipid bilayer. Second, protein-based sorting machinery generates transport carriers capable of concentrating specific cargo proteins and targeting to appropriate acceptor membranes, where they fuse and deliver their cargo.

Protein Sorting by the Lipid Gradient across the Secretory Membrane System

A conserved feature of the secretory membrane system is the differential distribution of various classes of lipids along the pathway. These classes of lipids include glycerophospholipids (phosphoglycerides), sphingolipids (e.g., sphingomyelin and glycosphingolipids), and cholesterol (see Figs. 7-4 and 20-13). These lipids play a major role in the sorting of proteins within the secretory membrane system because of their immiscibility (i.e., the property of not mixing) in membranes with different lipid compositions. By not mixing with some lipids while mixing with others, these lipid classes form lateral lipid assemblies, termed microdomains, that can concentrate or exclude specific membrane proteins.

Studies using artificial membranes have demonstrated how lipid immiscibility allows a continuous lipid bilayer to self-organize into distinct lipid domains with unique lipid compositions and biophysical properties. A prime example is an artificial bilayer containing glycerophospholipids and cholesterol to which sphingolipid is added; after sphingolipid is added, the cholesterol and glycerophospholipids partition into distinct domains (Fig. 21-3A–B). Because of van der Waals attraction between the sphingolipid’s long, saturated hydrocarbon chain and cholesterol’s rigid, flat-cylindrical steroid backbone, the cholesterol and sphingolipids associate in the plane of the membrane, whereas glycerophospholipids, which have unsaturated, kinked hydrocarbon chains with much less affinity for cholesterol, are largely excluded from the cholesterol/sphingolipid domains. The domains enriched in cholesterol/sphingolipid are thicker than the surrounding membrane composed of shorter, unsaturated, kinked glycerophospholipids (Fig. 21-3C). Tension on the bilayer (i.e., from binding of proteins that bend or curve the membrane) enhances the tendency of lipids that have different physical properties to separate into distinct phases.

Figure 21-3 properties of lipids within membranes. a–b, Cartoon depiction of an artificial bilayer containing a POPC/cholesterol mixture of 2:1 (A) and a POPC/cholesterol/sphingomyelin mixture of 2:1:1 (POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) (B). The blue spherical spots in part B are cholesterol- and sphingomyelin-enriched domains that have segregated from POPC because of the affinity between sphingomyelin and cholesterol. C, A bilayer enriched in cholesterol and sphingomyelin has a greater thickness than a bilayer composed mainly of glycerophospholipids, owing to the long saturated hydrocarbon chains of glycosphingolipids that attract proteins with longer transmembrane domains. D, Glycerophospholipid-containing membranes with high concentrations of cholesterol have a greater thickness than those with low concentrations, owing to a tighter alignment of the hydrocarbon chains. Scale bar is 5 μm.

In addition to prompting separation of sphingolipids from glycerophospholipids, cholesterol can affect a bilayer composed of glycerophospholipids alone (Fig. 21-3 D). In this case, the cholesterol fills the space between the floppy hydrocarbon chains of glycerophospholipids in the bilayer. This forces the glycerophospholipids into a tighter alignment and increases the distance between their head groups. As a result, the bilayer becomes thicker, resembling the thickness of bilayers enriched in sphingomyelin alone or sphingomyelin plus cholesterol.

Sphingolipids (e.g., glycosphingolipids and sphingomyelin) are synthesized in the Golgi apparatus, while the ER produces cholesterol and glycerophospholipids. Synthesis of these lipids at two different sites, combined with the self-organizing capacity of sphingolipids, cholesterol, and glycerophospholipids, gives rise to a pattern of lipid circulation within the secretory system that plays important roles in membrane sorting (Fig. 21-4A). Newly synthesized cholesterol is continually removed from the ER and redistributed to the Golgi apparatus, where high affinity interactions with sphingolipids prevent it from returning to the ER. The association of cholesterol with sphingolipids in the Golgi apparatus, in turn, triggers the lateral differentiation of domains enriched in these lipids. Through the additional activity of protein-based sorting and trafficking machinery, these domains bud off the Golgi apparatus and move to the plasma membrane, redistributing sphingolipids and cholesterol to the cell surface.

Figure 21-4 a lipid gradient arises across the secretory pathway as a result of the self-organizing properties of glycerophospholipids, sphingolipids, and cholesterol and their differential sites of synthesis. The gradient helps to sort and transport proteins to different sites within the secretory system. A, Lipid circulation and sorting within the secretory membrane system. Glycerophospholipids (GPL) and cholesterol (sterol) are synthesized in the ER, whereas sphingolipids (SL), including sphingomyelin and glycosphingolipids, are synthesized in the Golgi apparatus. Cholesterol that moves to the Golgi from the ER associates with SL and is carried to the plasma membrane. This gives rise to different concentrations of these lipids in these organelles at steady state and results in lipid environments in the ER and plasma membrane that are compatible with their functions (e.g., protein translocation for the ER and low permeability for the plasma membrane). B–D, Sorting of transmembrane proteins based on the length of their transmembrane domains. The distinct lipid compositions of the ER, Golgi apparatus, and plasma membrane result in bilayers that differ in thickness (with the ER bilayer depleted of SL/sterols and thin, the plasma membrane bilayer enriched in SL/sterols and thick, and the Golgi bilayer intermediate in SL/sterol content and having mixed thickness). To avoid hydrophobic mismatch, transmembrane proteins move to the organelle whose bilayer thickness best matches that of the protein’s transmembrane domain length.

The forward flow of cholesterol, sphingolipids, and glycerophospholipids toward the plasma membrane is balanced by selective retrograde flow. Glycerophospholipids transferred from the ER to the Golgi apparatus are recycled back to the ER. Similarly, sphingolipids delivered to the plasma membrane from the Golgi apparatus are returned to the Golgi apparatus. Cholesterol, in contrast, is not returned through these retrograde pathways to either the ER or the Golgi apparatus but enters and circulates within the endocytic pathway leading to lysosomes. This pattern of lipid circulation creates a gradient of cholesterol, sphingolipids, and glycerophospholipids across the secretory membrane system. Within this gradient, the ER has a low concentration of cholesterol (e.g., sterols) and sphingolipids, the Golgi apparatus has an intermediate concentration, and the plasma membrane has a high concentration (Fig. 21-4 A).

The lipid gradient serves two important functions. First, it generates different lipid environments in the ER, Golgi apparatus, and plasma membrane compatible with their distinct functions. The low concentration of sterols and sphingolipids in the ER membrane means that it is composed primarily of glycerophospholipids (i.e., phosphatidylcholine, PC; phosphatidylserine, PS; and phosphatidylethanolamine, PE). The loosely packed acyl chains of PC, PS, and PE are readily deformable, permitting newly synthesized membrane proteins to insert into and fold in the ER bilayer. This feature explains why the ER is used as the sole site of cotranslational protein synthesis in the cell. By contrast, the high concentration of sterols and sphingolipids makes the plasma membrane bilayer thicker and less permeable to small molecules. This allows the plasma membrane to form a flexible but impermeable barrier between the cytoplasm and cell exterior. The intermediate concentration of sterols and sphingolipids in the Golgi apparatus allows it to serve as a membrane-sorting station.

A second function of the lipid gradient is to promote sorting of transmembrane proteins within the secretory system. Each integral membrane protein seeks a lipid bilayer with a thickness that matches the lengths of its transmembrane segments (Fig. 21-4B–D). Because most transmembrane segments are stiff hydrophobic a-helices, it is energetically unfavorable to expose hydrophobic residues of a transmembrane polypeptide to the aqueous environment of the cytoplasm or vesicle lumen or to bury hydrophilic amino acids with the lipid acyl chains in the interior of the membrane. To avoid such hydrophobic mismatches, integral membrane proteins of the secretory system have evolved with transmembrane segments that are matched to the thickness of their target membranes. Hence, resident membrane proteins in the ER and Golgi apparatus typically have shorter transmembrane segments (around 15 amino acids) than do resident plasma membrane proteins (approximately 20 to 25 amino acids). Retention and/or transport of these proteins occurs because the lipid bilayers of carriers budding out from either the ER (toward the Golgi apparatus) or the Golgi apparatus (toward the plasma membrane) are thicker than the bilayers of the donor organelles. Only transmembrane proteins with transmembrane segments long enough to span this thickness enter such carriers.

If the transmembrane segment of a plasma membrane protein is shortened experimentally by using recombinant DNA techniques, the new protein is retained in the thinner bilayers of the ER and/or Golgi apparatus rather than moving on to the thicker plasma membrane. Similarly, when the transmembrane segment of a Golgi protein is extended, the protein is no longer retained in the Golgi apparatus but is transported to the plasma membrane.

This lipid-based protein sorting mechanism takes advantage of the lipid gradient established by the self-organizing properties of glycerophospholipids, cholesterol, and sphingolipids to sort and transport proteins within the secretory system. It is not, however, the only mechanism used by cells to organize and transport proteins along the secretory pathway. In addition, a complex protein-based machinery is relied on to bring far greater specificity and efficiency to these processes.

Protein-Based Machinery for Protein Sorting and Transport within the Secretory Membrane System

Sorting and transporting proteins within the secretory membrane system depend on several types of proteins (Fig. 21-5): Specialized “coats” help to generate both small and large transport carriers and sort proteins into them; motor proteins move carriers along the cytoskeleton; “tethering factors” attach carriers to the cytoskeleton and to their destination organelles prior to fusion; and fusion proteins mediate fusion of the carrier with an acceptor membrane. These components also associate with specific organelles, providing organelles with an identity that is both unique and dynamic. Many of the components are peripheral membrane proteins that lack transmembrane domains, so they must be recruited to the cytoplasmic surface of appropriate membranes by binding to either specific lipids, such as phosphoinositides, or to activated GTPases. Cells regulate the distributions of these organelle-specific lipids and GTPases. When infectious agents or stressful conditions disrupt these targeting molecules, secretory membrane trafficking can be disorganized and/or inhibited. The following sections describe the six major protein-based mechanisms that are used for sorting, transport, and fusion in the secretory membrane system.

Figure 21-5 protein machinery for secretory transport. Coat proteins that cluster into polymerized arrays help to sort soluble cargo and transmembrane proteins into a coated bud that pinches off a donor membrane as a coated vesicle (A) or larger vesicular tubular carriers (B). The carriers move by motor proteins along either microtubules or actin. Tethering factors, including long coiled-coil proteins or multimeric tethering complexes, tether the carriers to an acceptor membrane. SNARE proteins on the carrier and acceptor membrane then form a complex that drives membrane fusion, leading to delivery of the carrier’s content to the acceptor membrane.

Arf GTPases

The Arf family of GTPases includes Sar1, Arf1-6 and several distantly related Arf-like GTPases. These small GTPases mediate the association of a wide variety of protein effectors with specific membranes, which, in turn, leads to the differentiation of membrane domains that give rise to transport carriers and create compartmental identity.

Like other GTPases (see Figs. 4-6 and 4-7), Arfs are molecular switches that alternate between a GTP-bound active form that interacts with effector targets and a GDP-bound inactive form that does not (Fig. 21-6). Active Arf GTPases associate with membranes, where-as inactive GTPases are cytoplasmic. Specific GTP exchange factors (GEFs) recruit Arf proteins to particular membrane surfaces and then catalyze the exchange of GDP for GTP. When associated with particular membranes active Arfs bind their effectors until a GTPase-activating protein (GAP) induces hydrolysis of GTP, reversing membrane association and effector binding. The distribution of GEFs on particular membranes determines the location of specific active Arfs. Similarly, the location of GAPs determines where each type of Arf is inactivated.

Figure 21-6 Arf-GTPase cycle. A–C, Ribbon diagrams of Arf1-GDP (A), Arf1-GTP (B), and free Arf1 and Arf1 bound to its GAP (C). D, Membrane binding and dissociation of Arf1. In the cytoplasm, Arf1 exists in its GDP-bound form with its N-terminal amphipathic helix tucked into a hydrophobic pocket. An N-terminal myristoyl group allows Arf1 to reversibly bind to membranes for activation by a GEF. The exchange of GDP for GTP induces a conformational change in switch 1 and 2, as well as in the interswitch loop, which displaces the N-terminal helix out of its pocket. This causes Arf1-GTP to bind tightly to membranes, since both the hydrophobic residues of the N-terminal helix and the myristoyl anchor associate with the bilayer. Arf1-GTP then recruits effectors. Association of a GAP with the Arf1-GTP-effector complex stimulates GTP hydrolysis. Arf1-GDP returns to the cytoplasm, and GAP and effector proteins dissociate from the membrane. Note that GDP-bound Arf1 has its N-terminal amphipathic helix (striped blue and pink) retracted into a hydrophobic pocket and its interswitch region (purple) retracted. The N-terminal myristoyl group (green) is still free to associate with membrane, but the binding is weak, resulting in reversible binding. On exchange of GDP for GTP, the switch 1 and 2 domains move, and the interswitch toggles out of the hydrophobic pocket, allowing tighter membrane binding. The drug BFA interferes with exchange of GDP for GTP on Arf1 by stabilizing the association between Arf1-GDP and its GEF. As a result, Arf1 cannot recruit effectors to the membrane, leading to disruption of membrane traffic between the ER and Golgi apparatus.

Activation of Arfs by exchange of GDP for GTP not only creates a binding site for target proteins (i.e., effectors) but also promotes interaction with the lipid bilayer. A myristoyl group covalently bound to the N-terminus of most Arfs allows them to interact transiently and nonspecifically with membranes. When a specific Arf-GEF on a membrane catalyzes the exchange of GDP for GTP, an amphipathic (hydrophobic on one side, hydrophilic on the other) N-terminal, a-helix is released from a hydrophobic pocket on the GTPase so that the hydrophobic side of the helix can interact with the bilayer (Fig. 21-6 D). The membrane-associated GEFs that are responsible for activating Arfs all contain an evolutionarily conserved domain (referred to as the Sec7 domain). Association of this domain with Arf1-GDP is stabilized in the presence of the toxic fungal metabolite brefeldin A (BFA [Fig. 21-6 D]). This prevents Arf1 conversion to its active, GTP-bound state and thereby blocks Arf1 activity, similar to that of a GDP-locked Arf1 mutant.

Arf GTPases of the secretory pathway, in particular Sar1 and Arf1, recruit to membranes many types of effector proteins. These include the coat protein complexes of COPII, COPI, and clathrin/adapters plus other effectors such as phospholipid modifiers (e.g., phospholipase D, a lipid metabolizing enzyme), phosphoinositides, and cytoskeletal components. The coat protein complexes assemble into large polymeric structures (called protein coats) at the cytoplasmic surface of ER, pre-Golgi, and Golgi membranes, from which they sort cargo and promote the budding of transport carriers. The other Arf effectors play roles in differentiating the membrane environment of these carriers and enabling them to move to different locations within the cell. The four other mammalian Arf proteins (Arfs 2 to 6) regulate vesicle formation at other locales in the exocytic and endocytic pathways.

Sar1 assembles the COPII coat complex that is involved in differentiating ER export domains, which are the sites from which transport carriers bud out from the ER. Arf1, by contrast, assembles the COPI coat complex that is involved in the creation of retrograde transport carriers that bud from pre-Golgi and Golgi structures. Arf1 also recruits the clathrin/adapter coat complexes that are involved in budding of transport carriers from the Golgi en route to the endosome/lysosomal system. Disruption of the GTPase cycles of either Sar1 or Arf1 has dramatic consequences for secretory transport and the organization of the secretory pathway (Fig. 21-14). When the GTPase cycles of Sar1 or Arf1 are disrupted, the Golgi apparatus disassembles, and Golgi enzymes return to the ER or to ER exit sites with all secretory transport out of the ER inhibited.

Figure 21-14 transport from the endoplasmic reticulum to the golgi apparatus. ER to Golgi transport is orchestrated by the combined activities of many molecules. Sar1 and its effectors initiate COPII-coated bud formation and clustering of cargo at regions called ER export domains. This induces p115 and Rab1 to bind to these regions, which in turn recruits GBF1, the GEF for Arf1. Subsequent recruitment of Arf1 and its effectors further differentiates the ER export domain into a VTC. The VTC detaches from the ER and targets the Golgi apparatus, where it fuses with the cis face of the Golgi. The cargo in the VTC is then released into the Golgi and moves to the trans Golgi (where it will exit from the TGN). Expression of a constitutively inactive Sar1 mutant, Sar1[T39N], blocks COPII recruitment, and no ER exit sites form. Expression of an inactive Arf1 mutant, Arf1[T31N], or BFA treatment blocks recruitment of Arf1 effectors, which prevents ER exit sites from differentiating into VTCs. This causes the shrinkage and disappearance of the Golgi apparatus because new membrane from the ER cannot be delivered to the Golgi.

The COPII Coat

The COPII coat complex (Fig. 21-7) is essential for sorting and trafficking secretory cargo out of the ER. It consists of Sar1p GTPase, the Sec23p˙Sec24p subcomplex, and the Sec13p˙Sec31p subcomplex. These components self-assemble into a polymeric, two-dimensional scaffold (called a coat) that then collects specific types of cargo. The intrinsic curvature of the coat promotes the formation of membrane buds that are capable of pinching off the membrane as coated vesicles.

Figure 21-7 copii coat assembly on endoplasmic reticulum membrane. a–b, Ribbon diagrams of Sar1-GDP (PDB file: 1F6B) and the Sec23p-24p complex with Sec24p bound to Sar1-GTP. C, The bow-tie structure of Sec23p-Sec24p provides an extensive membrane-interaction surface that is concave, positively charged, and suitable for curving the bilayer when the subcomplex is bound to a membrane surface. D, Electron micrograph of a thin section illustrates the formation of a typical COPII vesicle when ER membranes are incubated in a test tube with cytosol and ATP. E, Sec12p activates Sar1 by promoting exchange of GDP for GTP, bringing Sar1 to the membrane. Sar1p-GTP then recruits the Sec23p˙Sec24p subcomplex. Binding of Sec13p˙Sec31p to Sec23p˙Sec24p clusters these complexes into a coat. Transmembrane cargo is recruited into the coat by binding to Sec24p. Coat complexes dissociate from the lattice after Sar1-GTP converts to Sar1-GDP and releases into the cytosol. As long as coat oligomerization occurs faster than Sar1-dependent coat complex release, the lattice grows into a coated bud that can pinch off the membrane as a coated vesicle. Coat disassembly on the coated vesicle results from continued Sar1-dependent coat complex release in the absence of further coat complex addition due to Sec12 not being packaged into the coated vesicle membrane. F, Three-dimensional reconstruction of COPII cage at 30-å resolution using cryoelectron microscopy and single-particle analysis.

(C, Adapted from Bickford LC, Mossessova E, Goldberg J: A structural view of the COPII vesicle coat. Curr Opin Struct Biol 14:147–153, 2004, with permission from Elsevier. D, Courtesy of W. Balch, Scripps Research Institute, La Jolla, California. F, Adapted by permission from Macmillan Publishers Ltd. from Stagg SM, Gurkan C, Fowler DM, et al: Structure of the Sec13/31 COPII cage. Nature 439:234–238, 2006. Copyright 2006.)

COPII coats assemble by a sequential process (Fig. 21-7 E

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