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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. Many proteins involved at different steps in vesicle transport contain domains 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). phosphoinositide PI(4,5)P2 endocytosis signals AP2 open AP2 locked ˜2 µ2 ˛2 ˝


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Cell_Biology_Alberts. phosphoinositide PI(4,5)P2 endocytosis signals AP2 open AP2 locked ˜2 µ2 ˛2 ˝ 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.


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Cell_Biology_Alberts. 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. OHOHHOHOHOPOOOOOOCCOO_CH2CH2CH123456123456PIPI(3,4)P2PI(3,5)P2PI(3)PPI(3,4)P2PIPI(4)PPI(5)PPI(4,5)P2PI(3,4,5)P3(A)(C)(E)(F)(B)(D)PPPP Membrane-bending proteins help deform the Membrane during vesicle formation


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Cell_Biology_Alberts. The forces generated by clathrin coat assembly alone are not sufficient to shape and pinch off a vesicle from the membrane. Other membrane-bending 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


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. 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.)


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Cell_Biology_Alberts. M.a. de Matteis and a. godi, nat. Cell Biol. 6:487–492, 2004. with permission from Macmillan publishers ltd.) 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.


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Cell_Biology_Alberts. (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


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Cell_Biology_Alberts. M.a. de Matteis and a. godi, nat. Cell Biol. 6:487–492, 2004. with permission from Macmillan publishers ltd.) constitutive exocytosis regulated exocytosis endocytosis phagocytosis 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 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


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Cell_Biology_Alberts. The pinching-off process brings the two noncytosolic leaflets of the membrane proteins contain a distinctive curvature and into close proximity and fuses them, sealing off the forming vesicle (see Figure often have additional features that adapt them to their specific tasks: some have 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 distorting the bilayer structure, or by changing its lipid composition through the others are flanked by pIp-binding domains recruitment of lipid-modifying enzymes, or by both mechanisms. that direct them to membranes enriched in Once released from the membrane, the vesicle rapidly loses its clathrin coat. A cognate phosphoinositides. 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)


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Cell_Biology_Alberts. 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).


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GTPase domain of dynamin

Cell_Biology_Alberts. GTPase domain of dynamin


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Cell_Biology_Alberts. 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


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Cell_Biology_Alberts. 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 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.)


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. 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


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. 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


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. Clathrinand 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. inactive, soluble Sar1-GDP (A) amphiphilic helix active, membrane-bound Sar1-GTP donor membrane (ER) Sar1-GEF GTPGTPGDPGDPCYTOSOL ER LUMEN not all Transport vesicles are spherical


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Cell_Biology_Alberts. 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 rims of the 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.


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Cell_Biology_Alberts. 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


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. Figure 13–14 Formation of a COPII-coated 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.


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Cell_Biology_Alberts. (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.)


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Cell_Biology_Alberts. 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 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


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. 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.


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Cell_Biology_Alberts. 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


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Cell_Biology_Alberts. 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.) cytosol; in their GTP-bound state, they are active and tightly associated with the membrane of an organelle or transport vesicle. Membrane-bound Rab-GEFs activate Rab proteins on both transport vesicle and target membranes; for some membrane fusion events, activated Rab molecules are required on both sides of the reaction. Once in the GTP-bound state and membrane-bound through a now-exposed lipid anchor, Rab proteins bind to other proteins, called Rab effectors, which are the downstream mediators of vesicle transport, membrane tethering, and membrane fusion (Figure 13–16). The rate of GTP hydrolysis sets the concentration of active Rab and, consequently, the concentration of its effectors on the membrane.


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Cell_Biology_Alberts. In contrast to the highly conserved structure of Rab proteins, the structures and functions of Rab effectors vary greatly, and the same Rab proteins can often bind to many different effectors. Some Rab effectors are motor proteins that propel vesicles along actin filaments or microtubules to their target membrane. Others are tethering proteins, some of which have long, threadlike domains that serve as “fishing lines” that can extend to link two membranes more than 200 nm apart; other tethering proteins are large protein complexes that link two membranes that are closer together and interact with a wide variety of other proteins that facilitate the membrane fusion step. The tethering complex that docks COPII-coated


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Cell_Biology_Alberts. Figure 13–16 Tethering of a transport vesicle to a target membrane. rab effector proteins interact with active rab proteins (rab-gTps, yellow) located on the target membrane, vesicle membrane, or both, to establish the first connection between the two membranes that are going to fuse. In the example shown here, the rab effector is a filamentous tethering protein (dark green). next, snare proteins on the two membranes (red and blue) pair, docking the vesicle to the target membrane and catalyzing the fusion of the two apposed lipid bilayers. during docking and fusion, a rab-gap (not shown) induces the rab protein to hydrolyze its bound gTp to gdp, causing the rab to dissociate from the membrane and return to the cytosol as rab-gdp, where it is bound by a gdI protein that keeps the rab soluble and inactive.


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Cell_Biology_Alberts. vesicles, for example, contains a protein kinase that phosphorylates the coat proteins to complete the uncoating process. Coupling uncoating to vesicle delivery helps to ensure directionality of the transport process and fusion with the proper membrane. Rab effectors can also interact with SNAREs to couple membrane tethering to fusion (see Figure 13–16). The assembly of Rab proteins and their effectors on a membrane is cooperative and results in the formation of large, specialized membrane patches. Rab5, for example, assembles on endosomes and mediates the capture of endocytic vesicles arriving from the plasma membrane. The experimental depletion of Rab5 causes disappearance of the entire endosomal and lysosomal membrane system, highlighting the crucial role of Rab proteins in organelle biogenesis and maintenance.


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Cell_Biology_Alberts. A Rab5 domain concentrates tethering proteins that catch incoming vesicles. Its assembly on endosomal membranes begins when a Rab5-GDP/GDI complex encounters a Rab-GEF. GDI is released and Rab5-GDP is converted to Rab5-GTP. Active Rab5-GTP becomes anchored to the membrane and recruits more Rab5GEF to the endosome, thereby stimulating the recruitment of more Rab5 to the same site. In addition, active Rab5 activates a PI 3-kinase, which locally converts PI to PI(3)P, which in turn binds some of the Rab effectors including tethering proteins and stabilizes their local membrane attachment (Figure 13–17). This type of positive feedback greatly amplifies the assembly process and helps to establish functionally distinct membrane domains within a continuous membrane.


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Cell_Biology_Alberts. The endosomal membrane provides a striking example of how different Rab proteins and their effectors help to create multiple specialized membrane domains, each fulfilling a particular set of functions. Thus, while the Rab5 membrane domain receives incoming endocytic vesicles from the plasma membrane, distinct Rab11 and Rab4 domains in the same membrane organize the budding of recycling vesicles that return proteins from the endosome to the plasma membrane. rab cascades can change the Identity of an organelle


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Cell_Biology_Alberts. A Rab domain can be disassembled and replaced by a different Rab domain, changing the identity of an organelle. Such ordered recruitment of sequentially acting Rab proteins is called a Rab cascade. Over time, for example, Rab5 domains are replaced by Rab7 domains on endosomal membranes. This converts an early endosome, marked by Rab5, into a late endosome, marked by Rab7. Because the set of Rab effectors recruited by Rab7 is different from that recruited by Rab5, this change reprograms the compartment: as we discuss later, it alters the membrane dynamics, including the incoming and outgoing traffic, and repositions the organelle away from the plasma membrane toward the cell interior. All of the cargo contained in the early endosome that has not been recycled to the plasma membrane is now part of a late endosome. This process is also referred to as endosome maturation. The self-amplifying nature of the Rab domains renders the process of endosome maturation unidirectional and


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Cell_Biology_Alberts. is now part of a late endosome. This process is also referred to as endosome maturation. The self-amplifying nature of the Rab domains renders the process of endosome maturation unidirectional and irreversible (Figure 13–18).


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Cell_Biology_Alberts. Figure 13–17 The formation of a Rab5 domain on the endosome membrane. a rab5-gef on the endosome membrane binds a rab5 protein and induces it to exchange gdp for gTp. gdI is lost and gTp binding alters the conformation of the rab protein, exposing an amphiphilic helix and a covalently attached lipid group, which together anchor the rab5-gTp to the membrane. active rab5 activates pI 3-kinase, which converts pI into pI(3) p. pI(3)p and active rab5 together bind a variety of rab effector proteins that contain pI(3)p-binding sites, including filamentous tethering proteins that catch incoming clathrin-coated endocytic vesicles from the plasma membrane. with the help of another effector, active rab5 also recruits more rab5-gef, further enhancing the assembly of the rab5 domain on the membrane.


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Cell_Biology_Alberts. controlled cycles of gTp hydrolysis and gdp–gTp exchange dynamically regulate the size and activity of such rab domains. unlike snares, which are integral membrane proteins, the gdp/ gTp cycle, coupled to the membrane/ cytosol translocation cycle, endows the rab machinery with the ability to undergo assembly and disassembly on the membrane. (adapted from M. Zerial and h. Mcbride, nat. rev. Mol. Cell Biol. 2:107–117, 2001. with permission from Macmillan publishers ltd.)


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Cell_Biology_Alberts. Once a transport vesicle has been tethered to its target membrane, it unloads its cargo by membrane fusion. Membrane fusion requires bringing the lipid bilayers of two membranes to within 1.5 nm of each other so that they can merge. When the membranes are in such close apposition, lipids can flow from one bilayer to the other. For this close approach, water must be displaced from the hydrophilic surface of the membrane—a process that is highly energetically unfavorable and requires specialized fusion proteins that overcome this energy barrier. We have already discussed the role of dynamin in a related task during the pinching-off of clathrin-coated vesicles (see Figure 13–13).


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Cell_Biology_Alberts. The SNARE proteins (also called SNAREs, for short) catalyze the membrane fusion reactions in vesicle transport. There are at least 35 different SNAREs in an animal cell, each associated with a particular organelle in the secretory or endocytic pathways. These transmembrane proteins exist as complementary sets, with v-SNAREs usually found on vesicle membranes and t-SNAREs usually found on target membranes (see Figure 13–16). A v-SNARE is a single polypeptide chain, whereas a t-SNARE is usually composed of three proteins. The v-SNAREs and t-SNAREs have characteristic helical domains, and when a v-SNARE interacts with a t-SNARE, the helical domains of one wrap around the helical domains of the other to form a very stable four-helix bundle. The resulting trans-SNARE complex locks the two membranes together. Biochemical membrane fusion assays with all different SNARE combinations show that vand t-SNARE pairing is highly specific. The SNAREs thus provide an additional layer of specificity


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Cell_Biology_Alberts. together. Biochemical membrane fusion assays with all different SNARE combinations show that vand t-SNARE pairing is highly specific. The SNAREs thus provide an additional layer of specificity in the transport process by helping to ensure that vesicles fuse only with the correct target membrane.


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Cell_Biology_Alberts. The trans-SNARE complexes catalyze membrane fusion by using the energy that is freed when the interacting helices wrap around each other to pull the membrane faces together, simultaneously squeezing out water molecules from the interface (Figure 13–19). When liposomes containing purified v-SNAREs are mixed with liposomes containing complementary t-SNAREs, their membranes fuse, albeit slowly. In the cell, other proteins recruited to the fusion site, presumably Rab effectors, cooperate with SNAREs to accelerate fusion. Fusion does not always follow immediately after v-SNAREs and t-SNAREs pair. As we discuss later, in the process of regulated exocytosis, fusion is delayed until secretion is triggered by a specific extracellular signal.


Cell_Biology_Alberts

Cell_Biology_Alberts. Rab proteins, which can regulate the availability of SNARE proteins, exert an additional layer of control. t-SNAREs in target membranes are often associated with inhibitory proteins that must be released before the t-SNARE can function. Rab proteins and their effectors trigger the release of such SNARE inhibitory Figure 13–19 A model for how SNARE proteins may catalyze membrane fusion. bilayer fusion occurs in multiple steps. a tight pairing between vand t-snares forces lipid bilayers into close apposition and expels water molecules from the interface. lipid molecules in the two interacting (cytosolic) leaflets of the bilayers then flow between the membranes to form a connecting stalk. lipids of the two noncytosolic leaflets then contact each other, forming a new bilayer, which widens the fusion zone (hemifusion, or half-fusion). rupture of the new bilayer completes the fusion reaction.


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Cell_Biology_Alberts. Figure 13–18 A model for a generic Rab cascade. The local activation of a raba-gef leads to assembly of a raba domain on the membrane. active raba recruits its effector proteins, one of which is a gef for rabb. The rabb-gef then recruits rabb to the membrane, which in turn begins to recruit its effectors, among them a gap for raba. The raba-gap activates raba gTp hydrolysis leading to the inactivation of the raba and the disassembly of the raba domain as the rabb domain grows. In this way, the raba domain is irreversibly replaced by the rabb domain. In principle, this sequence can continued by the recruitment of a next gef by rabb. (adapted from a.h. hutagalung and p.J. novick, Physiol. rev. 91:119–149, 2011. with permission from The american physiological society.) proteins. In this way, SNARE proteins are concentrated and activated in the correct location on the membrane, where tethering proteins capture incoming vesicles. Rab proteins thus speed up the process by which appropriate


Cell_Biology_Alberts

Cell_Biology_Alberts. SNARE proteins are concentrated and activated in the correct location on the membrane, where tethering proteins capture incoming vesicles. Rab proteins thus speed up the process by which appropriate SNARE proteins in two membranes find each other.


Cell_Biology_Alberts

Cell_Biology_Alberts. For vesicle transport to operate normally, transport vesicles must incorporate the appropriate SNARE and Rab proteins. Not surprisingly, therefore, many transport vesicles will form only if they incorporate the appropriate complement of SNARE and Rab proteins in their membrane. How this crucial control process operates during vesicle budding remains a mystery. Interacting snares need to be pried apart before They can function again


Cell_Biology_Alberts

Cell_Biology_Alberts. Most SNARE proteins in cells have already participated in multiple rounds of vesicle transport and are sometimes present in a membrane as stable complexes with partner SNAREs. The complexes have to disassemble before the SNAREs can mediate new rounds of transport. A crucial protein called NSF cycles between membranes and the cytosol and catalyzes the disassembly process. NSF is a hexameric ATPase of the family of AAA-ATPases (see Figure 6–85) that uses the energy of ATP hydrolysis to unravel the intimate interactions between the helical domains of paired SNARE proteins (Figure 13–20). The requirement for NSF-mediated reactivation of SNAREs by SNARE complex disassembly helps prevent membranes from fusing indiscriminately: if the t-SNAREs in a target membrane were always active, any membrane containing an appropriate v-SNARE might fuse whenever the two membranes made contact. It is not known how the activity of NSF is controlled so that the SNARE machinery is activated at the right time


Cell_Biology_Alberts

Cell_Biology_Alberts. containing an appropriate v-SNARE might fuse whenever the two membranes made contact. It is not known how the activity of NSF is controlled so that the SNARE machinery is activated at the right time and place. It is also not known how v-SNAREs are selectively retrieved and returned to their compartment of origin so that they can be reused in newly formed transport vesicles.


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Cell_Biology_Alberts. Membrane fusion is important in other processes beside vesicle transport. The plasma membranes of a sperm and an egg fuse during fertilization, and myoblasts fuse with one another during the development of multinucleate muscle fibers (discussed in Chapter 22). Likewise, the ER network and mitochondria fuse and fragment in a dynamic way (discussed in Chapters 12 and 14). All cell membrane fusions require special proteins and are tightly regulated to ensure that only appropriate membranes fuse. The controls are crucial for maintaining both the identity of cells and the individuality of each type of intracellular compartment.


Cell_Biology_Alberts

Cell_Biology_Alberts. The membrane fusions catalyzed by viral fusion proteins are well understood. These proteins have a crucial role in permitting the entry of enveloped viruses (which have a lipid-bilayer-based membrane coat) into the cells that they infect (discussed in Chapters 5 and 23). For example, viruses such as the human immunodeficiency virus (HIV), which causes AIDS, bind to cell-surface receptors and then fuse with the plasma membrane of the target cell (Figure 13–21). This fusion event allows the viral nucleic acid inside the nucleocapsid to enter the cytosol, where it replicates. Other viruses, such as the influenza virus, first enter the cell by receptor-mediated endocytosis (discussed later) and are delivered to endosomes; the low pH in endosomes activates a fusion protein in the viral envelope that catalyzes the fusion of the viral and endosomal membranes, releasing the viral nucleic acid into the cytosol. Viral fusion proteins and SNAREs promote lipid bilayer fusion in similar ways.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 13–20 Dissociation of SNARE pairs by NSF after a membrane fusion cycle. after a v-snare and t-snare have mediated the fusion of a transport vesicle with a target membrane, nsf binds to the snare complex and, with the help of accessory proteins, hydrolyzes aTp to pry the snares apart. Directed and selective transport of particular membrane components from one membrane-enclosed compartment to another in a eukaryotic cell maintains the differences between those compartments. Transport vesicles, which can be spherical, tubular, or irregularly shaped, bud from specialized coated regions of the donor membrane. The assembly of the coat helps to collect specific membrane and soluble cargo molecules for transport and to drive the formation of the vesicle.


Cell_Biology_Alberts

Cell_Biology_Alberts. There are various types of coated vesicles. The best characterized are clathrin-coated vesicles, which mediate transport from the plasma membrane and the trans Golgi network, and COPIand COPII-coated vesicles, which mediate transport between Golgi cisternae and between the ER and the Golgi apparatus, respectively. Coats have a common two-layered structure: an inner layer formed of adaptor proteins links the outer layer (or cage) to the vesicle membrane and also traps specific cargo molecules for packaging into the vesicle. The coat is shed before the vesicle fuses with its appropriate target membrane.


Cell_Biology_Alberts

Cell_Biology_Alberts. Local synthesis of specific phosphoinositides creates binding sites that trigger clathrin coat assembly and vesicle budding. In addition, monomeric GTPases help regulate various steps in vesicle transport, including both vesicle budding and docking. The coat-recruitment GTPases, including Sar1 and the ARF proteins, regulate coat assembly and disassembly. A large family of Rab proteins functions as vesicle-targeting GTPases. Rab proteins are recruited to both, forming transport vesicles and target membranes. The assembly and disassembly of Rab proteins and their effectors in specialized membrane domains are dynamically controlled by GTP binding and hydrolysis. Active Rab proteins recruit Rab effectors, such as motor proteins, which transport vesicles along actin filaments or microtubules, and filamentous tethering proteins, which help ensure that the vesicles deliver their contents only to the appropriate target membrane. Complementary v-SNARE proteins on transport vesicles and t-SNARE


Cell_Biology_Alberts

Cell_Biology_Alberts. filamentous tethering proteins, which help ensure that the vesicles deliver their contents only to the appropriate target membrane. Complementary v-SNARE proteins on transport vesicles and t-SNARE proteins on the target membrane form stable trans-SNARE complexes, which force the two membranes into close apposition so that their lipid bilayers can fuse.


Cell_Biology_Alberts

Cell_Biology_Alberts. TransporT froM The er Through The golgI apparaTus As discussed in Chapter 12, newly synthesized proteins cross the ER membrane from the cytosol to enter the secretory pathway. During their subsequent transport, from the ER to the Golgi apparatus and from the Golgi apparatus to the cell surface and elsewhere, these proteins are successively modified as they pass through a series of compartments. Transfer from one compartment to the next involves a delicate balance between forward and backward (retrieval) transport pathways. Some transport vesicles select cargo molecules and move them to the next compartment in the pathway, while others retrieve escaped proteins and return them to a previous compartment where they normally function. Thus, the pathway from the ER to the cell surface consists of many sorting steps, which continuously select membrane and soluble lumenal proteins for packaging and transport.


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Cell_Biology_Alberts. Figure 13–21 The entry of enveloped viruses into cells. electron micrographs showing how hIv enters a cell by fusing its membrane with the plasma membrane of the cell. (from b.s. stein et al., Cell 49:659–668, 1987. with permission from elsevier.)


Cell_Biology_Alberts

Cell_Biology_Alberts. In this section, we focus mainly on the Golgi apparatus (also called the Golgi complex). It is a major site of carbohydrate synthesis, as well as a sorting and dispatching station for products of the ER. The cell makes many polysaccharides in the Golgi apparatus, including the pectin and hemicellulose of the cell wall in plants and most of the glycosaminoglycans of the extracellular matrix in animals (discussed in Chapter 19). The Golgi apparatus also lies on the exit route from the ER, and a large proportion of the carbohydrates that it makes are attached as oligosaccharide side chains to the many proteins and lipids that the ER sends to it. A subset of these oligosaccharide groups serve as tags to direct specific proteins into vesicles that then transport them to lysosomes. But most proteins and lipids, once they have acquired their appropriate oligosaccharides in the Golgi apparatus, are recognized in other ways for targeting into the transport vesicles going to other destinations.


Cell_Biology_Alberts

Cell_Biology_Alberts. proteins leave the er in copII-coated Transport vesicles To initiate their journey along the secretory pathway, proteins that have entered the ER and are destined for the Golgi apparatus or beyond are first packaged into COPII-coated transport vesicles. These vesicles bud from specialized regions of the ER called ER exit sites, whose membrane lacks bound ribosomes. Most animal cells have ER exit sites dispersed throughout the ER network.


Cell_Biology_Alberts

Cell_Biology_Alberts. Entry into vesicles that leave the ER can be a selective process or can happen by default. Many membrane proteins are actively recruited into such vesicles, where they become concentrated. These cargo membrane proteins display exit (transport) signals on their cytosolic surface that adaptor proteins of the inner COPII coat recognize (Figure 13–22); some of these components act as cargo receptors and are recycled back to the ER after they have delivered their cargo to the Golgi apparatus. Soluble cargo proteins in the ER lumen, by contrast, have exit signals that attach them to transmembrane cargo receptors. Proteins without exit signals can also enter transport vesicles, including protein molecules that normally function in the ER (so-called ER resident proteins), some of which slowly leak out of the ER and are delivered to the Golgi apparatus. Different cargo proteins enter the transport vesicles with substantially different rates and efficiencies, which may result from differences


Cell_Biology_Alberts

Cell_Biology_Alberts. out of the ER and are delivered to the Golgi apparatus. Different cargo proteins enter the transport vesicles with substantially different rates and efficiencies, which may result from differences in their folding and oligomerization efficiencies and kinetics, as well as the factors already discussed. The exit step from the ER is a major checkpoint at which quality control is exerted on the proteins that a cell secretes or displays on its surface, as we discussed in Chapter 12.


Cell_Biology_Alberts

Cell_Biology_Alberts. The exit signals that direct soluble proteins out of the ER for transport to the Golgi apparatus and beyond are not well understood. Some transmembrane proteins that serve as cargo receptors for packaging some secretory proteins into COPII-coated vesicles are lectins that bind to oligosaccharides on the secreted


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 13–22 The recruitment of membrane and soluble cargo molecules into ER transport vesicles. Membrane proteins are packaged into budding transport vesicles through interactions of exit signals on their cytosolic tails with adaptor proteins of the inner copII coat. some of these membrane proteins function as cargo receptors, binding soluble proteins in the er lumen and helping to package them into vesicles. other proteins may enter the vesicle by bulk flow. a typical 50 nm transport vesicle contains about 200 membrane proteins, which can be of many different types. as indicated, unfolded or incompletely assembled proteins are bound to chaperones and transiently retained in the er compartment.


Cell_Biology_Alberts

Cell_Biology_Alberts. proteins. One such lectin, for example, binds to mannose on two secreted blood-clotting factors (Factor V and Factor VIII), thereby packaging the proteins into transport vesicles in the ER; its role in protein transport was identified because humans who lack it owing to an inherited mutation have lowered serum levels of Factors V and VIII, and they therefore bleed excessively. only proteins That are properly folded and assembled can leave the er


Cell_Biology_Alberts

Cell_Biology_Alberts. To exit from the ER, proteins must be properly folded and, if they are subunits of multiprotein complexes, they need to be completely assembled. Those that are misfolded or incompletely assembled transiently remain in the ER, where they are bound to chaperone proteins (discussed in Chapter 6) such as BiP or calnexin. The chaperones may cover up the exit signals or somehow anchor the proteins in the ER. Such failed proteins are eventually transported back into the cytosol, where they are degraded by proteasomes (discussed in Chapters 6 and 12). This quality-control step prevents the onward transport of misfolded or misassembled proteins that could potentially interfere with the functions of normal proteins. Such failures are surprisingly common. More than 90% of the newly synthesized subunits of the T cell receptor (discussed in Chapter 24) and of the acetylcholine receptor (discussed in Chapter 11), for example, are normally degraded without ever reaching the cell surface where they


Cell_Biology_Alberts

Cell_Biology_Alberts. of the T cell receptor (discussed in Chapter 24) and of the acetylcholine receptor (discussed in Chapter 11), for example, are normally degraded without ever reaching the cell surface where they function. Thus, cells must make a large excess of some protein molecules to produce a select few that fold, assemble, and function properly.


Cell_Biology_Alberts

Cell_Biology_Alberts. Sometimes, however, there are drawbacks to the stringent quality-control mechanism. The predominant mutations that cause cystic fibrosis, a common inherited disease, result in the production of a slightly misfolded form of a plasma membrane protein important for Cl– transport. Although the mutant protein would function normally if it reached the plasma membrane, it is retained in the ER and then is degraded by cytosolic proteasomes. This devastating disease thus results not because the mutation inactivates the protein but because the active protein is discarded before it reaches the plasma membrane. vesicular Tubular clusters Mediate Transport from the er to the golgi apparatus


Cell_Biology_Alberts

Cell_Biology_Alberts. vesicular Tubular clusters Mediate Transport from the er to the golgi apparatus After transport vesicles have budded from ER exit sites and have shed their coat, they begin to fuse with one another. The fusion of membranes from the same compartment is called homotypic fusion, to distinguish it from heterotypic fusion, in which a membrane from one compartment fuses with the membrane of a different compartment. As with heterotypic fusion, homotypic fusion requires a set of matching SNAREs. In this case, however, the interaction is symmetrical, with both membranes contributing v-SNAREs and t-SNAREs (Figure 13–23). The structures formed when ER-derived vesicles fuse with one another are called vesicular tubular clusters, because they have a convoluted appearance in


Cell_Biology_Alberts

Cell_Biology_Alberts. The structures formed when ER-derived vesicles fuse with one another are called vesicular tubular clusters, because they have a convoluted appearance in Figure 13–23 Homotypic membrane fusion. In step 1, nsf pries apart identical pairs of v-snares and t-snares in both membranes (see figure 13–20). In steps 2 and 3, the separated matching snares on adjacent identical membranes interact, which leads to membrane fusion and the formation of one continuous compartment. subsequently, the compartment grows by further homotypic fusion with vesicles from the same kind of membrane, displaying matching snares. homotypic fusion occurs when er-derived transport vesicles fuse with one another, but also when endosomes fuse to generate larger endosomes. rab proteins help regulate the extent of homotypic fusion and hence the size of a cell’s compartments (not shown).


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Cell_Biology_Alberts. Figure 13–24 Vesicular tubular clusters. (a) an electron micrograph of vesicular tubular clusters forming around an exit site. Many of the vesicle-like structures seen in the micrograph are cross sections of tubules that extend above and below the plane of this thin section and are interconnected. (b) vesicular tubular clusters move along microtubules to carry proteins from the ER er to the golgi apparatus. copI-coated vesicles mediate the budding of vesicles that return to the (A) 0.2 µm er from these clusters (and from the golgi apparatus). (a, courtesy of william balch.) the electron microscope (Figure 13–24A). These clusters constitute a compartment that is separate from the ER and lacks many of the proteins that function in the ER. They are generated continually and function as transport containers that bring material from the ER to the Golgi apparatus. The clusters move quickly along microtubules to the Golgi apparatus with which they fuse (Figure 13–24B and Movie 13.2).


Cell_Biology_Alberts

Cell_Biology_Alberts. As soon as vesicular tubular clusters form, they begin to bud off transport vesicles of their own. Unlike the COPII-coated vesicles that bud from the ER, these vesicles are COPI-coated (see Figure 13–24A). COPI-coated vesicles are unique in that the components that make up the inner and outer coat layers are recruited as a preassembled complex, called coatomer. They function as a retrieval pathway, carrying back ER resident proteins that have escaped, as well as proteins such as cargo receptors and SNAREs that participated in the ER budding and vesicle fusion reactions. This retrieval process demonstrates the exquisite control mechanisms that regulate coat assembly reactions. The COPI coat assembly begins only seconds after the COPII coats have been shed, and remains a mystery how this switch in coat assembly is controlled.


Cell_Biology_Alberts

Cell_Biology_Alberts. The retrieval (or retrograde) transport continues as the vesicular tubular clusters move toward the Golgi apparatus. Thus, the clusters continuously mature, gradually changing their composition as selected proteins are returned to the ER. The retrieval continues from the Golgi apparatus, after the vesicular tubular clusters have delivered their cargo. The retrieval pathway to the er uses sorting signals The retrieval pathway for returning escaped proteins back to the ER depends on ER retrieval signals. Resident ER membrane proteins, for example, contain signals that bind directly to COPI coats and are thus packaged into COPI-coated transport vesicles for retrograde delivery to the ER. The best-characterized retrieval signal of this type consists of two lysines, followed by any two other amino acids, at the extreme C-terminal end of the ER membrane protein. It is called a KKXX sequence, based on the single-letter amino acid code.


Cell_Biology_Alberts

Cell_Biology_Alberts. Soluble ER resident proteins, such as BiP, also contain a short ER retrieval signal at their C-terminal end, but it is different: it consists of a Lys-Asp-Glu-Leu or a similar sequence. If this signal (called the KDEL sequence) is removed from BiP by genetic engineering, the protein is slowly secreted from the cell. If the signal is transferred to a protein that is normally secreted, the protein is now efficiently returned to the ER, where it accumulates. FORWARD PATHWAY RETRIEVAL PATHWAY COPII coat COPI coat vesicular tubular cluster cis cis, medial, and trans


Cell_Biology_Alberts

Cell_Biology_Alberts. Unlike the retrieval signals on ER membrane proteins, which can interact directly with the COPI coat, soluble ER resident proteins must bind to specialized receptor proteins such as the KDEL receptor—a multipass transmembrane protein that binds to the KDEL sequence and packages any protein displaying it into COPI-coated retrograde transport vesicles (Figure 13–25). To accomplish this task, the KDEL receptor itself must cycle between the ER and the Golgi apparatus, and its affinity for the KDEL sequence must differ in these two compartments. The receptor must have a high affinity for the KDEL sequence in vesicular tubular clusters and the Golgi apparatus, so as to capture escaped, soluble ER resident proteins that are present there at low concentration. It must have a low affinity for the KDEL sequence in the ER, however, to unload its cargo in spite of the very high concentration of KDEL-containing soluble resident proteins in the ER.


Cell_Biology_Alberts

Cell_Biology_Alberts. How does the affinity of the KDEL receptor change depending on the compartment in which it resides? The answer is likely related to the lower pH in the Golgi compartments, which is regulated by H+ pumps. As we discuss later, pH-sensitive protein–protein interactions form the basis for many of the protein sorting steps in the cell. Most membrane proteins that function at the interface between the ER and Golgi apparatus, including vand t-SNAREs and some cargo receptors, also enter the retrieval pathway back to the ER. Many proteins are selectively retained in the compartments in which They function


Cell_Biology_Alberts

Cell_Biology_Alberts. The KDEL retrieval pathway only partly explains how ER resident proteins are maintained in the ER. As mentioned, cells that express genetically modified ER resident proteins, from which the KDEL sequence has been experimentally removed, secrete these proteins. But the rate of secretion is much slower than for a normal secretory protein. It seems that a mechanism that is independent of their KDEL signal normally retains ER resident proteins and that only those proteins that escape this retention mechanism are captured and returned via the KDEL receptor. A suggested retention mechanism is that ER resident proteins bind to one another, thus forming complexes that are too big to enter transport vesicles efficiently. Because ER resident proteins are present in the ER at very high concentrations (estimated to be millimolar), relatively low-affinity interactions would suffice to retain most of the proteins in such complexes.


Cell_Biology_Alberts

Cell_Biology_Alberts. Aggregation of proteins that function in the same compartment is a general mechanism that compartments use to organize and retain their resident proteins. Golgi enzymes that function together, for example, also bind to each other and are thereby restrained from entering transport vesicles leaving the Golgi apparatus.


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Cell_Biology_Alberts. Figure 13–25 Retrieval of soluble ER resident proteins. er resident proteins that escape from the er are returned by vesicle transport. (a) The kdel receptor present in both vesicular tubular clusters and the golgi apparatus captures the soluble er resident proteins and carries them in copI-coated transport vesicles back to the er. (recall that the copIcoated vesicles shed their coats as soon as they are formed.) upon binding its ligands in the tubular cluster or golgi, the kdel receptor may change conformation, so as to facilitate its recruitment into budding copI-coated vesicles. (b) The retrieval of er proteins begins in vesicular tubular clusters and continues from later parts of the golgi apparatus. In the environment of the er, the er resident proteins dissociate from the kdel receptor, which is then returned to the golgi apparatus for reuse. we discuss the different compartments of the golgi apparatus shortly. The golgi apparatus consists of an ordered series of compartments


Cell_Biology_Alberts

Cell_Biology_Alberts. Because it could be selectively visualized by silver stains, the Golgi apparatus was one of the first organelles described by early light microscopists. It consists of a collection of flattened, membrane-enclosed compartments called cisternae, that somewhat resemble a stack of pita breads. Each Golgi stack typically consists of four to six cisternae (Figure 13–26), although some unicellular flagellates can have more than 20. In animal cells, tubular connections between corresponding cisternae link many stacks, thus forming a single complex, which is usually located near the cell nucleus and close to the centrosome (Figure 13–27A). This localization depends on microtubules. If microtubules are experimentally depolymerized, the Golgi apparatus reorganizes into individual stacks that are found throughout the cytoplasm, adjacent to ER exit sites. Some cells, including most plant cells, have hundreds of individual Golgi stacks dispersed throughout the cytoplasm where they are typically


Cell_Biology_Alberts

Cell_Biology_Alberts. found throughout the cytoplasm, adjacent to ER exit sites. Some cells, including most plant cells, have hundreds of individual Golgi stacks dispersed throughout the cytoplasm where they are typically found adjacent to ER exit sites (Figure 13–27B).


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Cell_Biology_Alberts. Figure 13–26 The Golgi apparatus. Three-dimensional reconstruction from electron micrographs of the golgi apparatus in a secretory animal cell. The cis face of the golgi stack is that closest to the er. (b) a thin-section electron micrograph of an animal cell. In plant cells, the golgi apparatus is generally more distinct and more clearly separated from other intracellular membranes than in animal cells. (a, redrawn from a. rambourg and y. clermont, Eur. J. Cell Biol. 51:189–200, 1990. with permission from wissenschaftliche verlagsgesellschaft; b, courtesy of brij J. gupta.) Figure 13–27 Localization of the Golgi apparatus in animal and plant cells. The golgi apparatus in a cultured fibroblast stained with a fluorescent antibody that recognizes a golgi resident protein (bright orange). The golgi apparatus is polarized, facing the direction in which the cell was crawling before fixation.


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Cell_Biology_Alberts. The golgi apparatus in a plant cell that is expressing a fusion protein consisting of a resident golgi enzyme fused to green fluorescent protein. (a, courtesy of John henley and Mark Mcniven; b, courtesy of chris hawes.)


Cell_Biology_Alberts

Cell_Biology_Alberts. During their passage through the Golgi apparatus, transported molecules undergo an ordered series of covalent modifications. Each Golgi stack has two distinct faces: a cis face (or entry face) and a trans face (or exit face). Both cis and trans faces are closely associated with special compartments, each composed of a network of interconnected tubular and cisternal structures: the cis Golgi network (CGN) and the trans Golgi network (TGN), respectively. The CGN is a collection of fused vesicular tubular clusters arriving from the ER. Proteins and lipids enter the cis Golgi network and exit from the trans Golgi network, bound for the cell surface or another compartment. Both networks are important for protein sorting: proteins entering the CGN can either move onward in the Golgi apparatus or be returned to the ER. Similarly, proteins exiting from the TGN move onward and are sorted according to their next destination: endosomes, secretory vesicles, or the cell surface. They also can be


Cell_Biology_Alberts

Cell_Biology_Alberts. or be returned to the ER. Similarly, proteins exiting from the TGN move onward and are sorted according to their next destination: endosomes, secretory vesicles, or the cell surface. They also can be returned to an earlier compartment. Some membrane proteins are retained in the part of the Golgi apparatus where they function.


Cell_Biology_Alberts

Cell_Biology_Alberts. As described in Chapter 12, a single species of N-linked oligosaccharide is attached en bloc to many proteins in the ER and then trimmed while the protein is still in the ER. The oligosaccharide intermediates created by the trimming reactions serve to help proteins fold and to help transport misfolded proteins to the cytosol for degradation in proteasomes. Thus, they play an important role in controlling the quality of proteins exiting from the ER. Once these ER functions have been fulfilled, the cell reutilizes the oligosaccharides for new functions. This begins in the Golgi apparatus, which generates the heterogeneous oligosaccharide structures seen in mature proteins. After arrival in the CGN, proteins enter the first of the Golgi processing compartments (the cis Golgi cisternae). They then move to the next compartment (the medial cisternae) and finally to the trans cisternae, where glycosylation is completed. The lumen of the trans cisternae is thought to be continuous with the


Cell_Biology_Alberts

Cell_Biology_Alberts. then move to the next compartment (the medial cisternae) and finally to the trans cisternae, where glycosylation is completed. The lumen of the trans cisternae is thought to be continuous with the TGN, the place where proteins are segregated into different transport packages and dispatched to their final destinations.


Cell_Biology_Alberts

Cell_Biology_Alberts. The oligosaccharide processing steps occur in an organized sequence in the Golgi stack, with each cisterna containing a characteristic mixture of processing enzymes. Proteins are modified in successive stages as they move from cisterna to cisterna across the stack, so that the stack forms a multistage processing unit. Investigators discovered the functional differences between the cis, medial, and trans subdivisions of the Golgi apparatus by localizing the enzymes involved in processing N-linked oligosaccharides in distinct regions of the organelle, both by physical fractionation of the organelle and by labeling the enzymes in electron microscope sections with antibodies (Figure 13–28). The removal of mannose and the addition of N-acetylglucosamine, for example, occur in the cis and medial cisternae, while the addition of galactose and sialic acid occurs in the trans cisterna and trans Golgi network. Figure 13–29 summarizes the functional compartmentalization of the Golgi apparatus.


Cell_Biology_Alberts

Cell_Biology_Alberts. oligosaccharide chains are processed in the golgi apparatus Whereas the ER lumen is full of soluble lumenal resident proteins and enzymes, the resident proteins in the Golgi apparatus are all membrane bound, as the enzymatic reactions apparently occur entirely on membrane surfaces. All of the Golgi glycosidases and glycosyl transferases, for example, are single-pass transmembrane proteins, many of which are organized in multienzyme complexes. Figure 13–28 Molecular compartmentalization of the Golgi apparatus.


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Cell_Biology_Alberts. Figure 13–28 Molecular compartmentalization of the Golgi apparatus. a series of electron micrographs shows the golgi apparatus (a) unstained, (b) stained with osmium, which preferentially labels the cisternae of the cis compartment, and (c and d) stained to reveal the location of specific enzymes. nucleoside diphosphatase is found in the trans golgi cisternae (c), while acid phosphatase is found in the trans golgi network (d). note that usually more than one cisterna is stained. The enzymes are therefore thought to be highly enriched rather than precisely localized to a specific cisterna. (courtesy of daniel s. friend.) 1 µm


Cell_Biology_Alberts

Cell_Biology_Alberts. Two broad classes of N-linked oligosaccharides, the complex oligosaccharides and the high-mannose oligosaccharides, are attached to mammalian glycoproteins. Sometimes, both types are attached (in different places) to the same polypeptide chain. Complex oligosaccharides are generated when the original N-linked oligosaccharide added in the ER is trimmed and further sugars are added; by contrast, high-mannose oligosaccharides are trimmed but have no new sugars added to them in the Golgi apparatus (Figure 13–30). The sialic acids in the = N-acetylneuraminic acid (sialic acid, or NANA) (C) HIGH-MANNOSE OLIGOSACCHARIDE


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Cell_Biology_Alberts. Figure 13–30 The two main classes of asparagine-linked (N-linked) oligosaccharides found in mature mammalian glycoproteins. (a) both complex oligosaccharides and high-mannose oligosaccharides share a common core region derived from the original n-linked oligosaccharide added in the er (see figure 12–50) and typically containing two n-acetylglucosamines (glcnac) and three mannoses (Man). (b) each complex oligosaccharide consists of a core region, together with a terminal region that contains a variable number of copies of a special trisaccharide unit (n-acetylglucosamine–galactose–sialic acid) linked to the core mannoses. frequently, the terminal region is truncated and contains only glcnac and galactose (gal) or just glcnac. In addition, a fucose may be added, usually to the core glcnac attached to the asparagine (asn). Thus, although the steps of processing and subsequent sugar addition are rigidly ordered, complex oligosaccharides can be heterogeneous. Moreover, although the complex


Cell_Biology_Alberts

Cell_Biology_Alberts. to the asparagine (asn). Thus, although the steps of processing and subsequent sugar addition are rigidly ordered, complex oligosaccharides can be heterogeneous. Moreover, although the complex oligosaccharide shown has three terminal branches, two and four branches are also common, depending on the glycoprotein and the cell in which it is made. (c) high-mannose oligosaccharides are not trimmed back all the way to the core region and contain additional mannoses. hybrid oligosaccharides with one Man branch and one glcnac and gal branch are also found (not shown).


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Cell_Biology_Alberts. The three amino acids indicated in (a) constitute the sequence recognized by the oligosaccharyl transferase enzyme that adds the initial oligosaccharide to the protein. ser, serine; Thr, threonine; x, any amino acid, except proline. Figure 13–29 Oligosaccharide processing in Golgi compartments. The localization of each processing step shown was determined by a combination of techniques, including biochemical subfractionation of the golgi apparatus membranes and electron microscopy after staining with antibodies specific for some of the processing enzymes. processing enzymes are not restricted to a particular cisterna; instead, their distribution is graded across the stack, such that early-acting enzymes are present mostly in the cis golgi cisternae and later-acting enzymes are mostly in the trans golgi cisternae. Man, mannose; glcnac, n-acetylglucosamine; gal, galactose; nana, n-acetylneuraminic acid (sialic acid). KEY: = N-acetylneuraminic acid (sialic acid, or NANA)


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Cell_Biology_Alberts. Figure 13–31 Oligosaccharide processing in the ER and the Golgi apparatus. The processing pathway is highly ordered, so that each step shown depends on the previous one. step 1: processing begins in the er with the removal of the glucoses from the oligosaccharide initially transferred to the protein. Then a mannosidase in the er membrane removes a specific mannose. The remaining steps occur in the golgi stack. step 2: golgi mannosidase I removes three more mannoses. step 3: n-acetylglucosamine transferase I then adds an n-acetylglucosamine. step 4: Mannosidase II then removes two additional mannoses. This yields the final core of three mannoses that is present in a complex oligosaccharide. at this stage, the bond between the two n-acetylglucosamines in the core becomes resistant to attack by a highly specific endoglycosidase (Endo H). since all later structures in the pathway are also endo h-resistant, treatment with this enzyme is widely used to distinguish complex from high-mannose


Cell_Biology_Alberts

Cell_Biology_Alberts. by a highly specific endoglycosidase (Endo H). since all later structures in the pathway are also endo h-resistant, treatment with this enzyme is widely used to distinguish complex from high-mannose oligosaccharides. step 5: finally, as shown in figure 13–30, additional n-acetylglucosamines, galactoses, and sialic acids are added. These final steps in the synthesis of a complex oligosaccharide occur in the cisternal compartments of the golgi apparatus: three types of glycosyl transferase enzymes act sequentially, using sugar substrates that have been activated by linkage to the indicated nucleotide; the membranes of the golgi cisternae contain specific carrier proteins that allow each sugar nucleotide to enter in exchange for the nucleoside phosphates that are released after the sugar is attached to the protein on the lumenal face.


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Cell_Biology_Alberts. note that, as a biosynthetic organelle, the golgi apparatus differs from the er: all sugars in the golgi are assembled inside the lumen from sugar nucleotide, whereas in the er, the n-linked precursor oligosaccharide is assembled partly in the cytosol and partly in the lumen, and most lumenal reactions use dolichol-linked sugars as their substrates (see figure 12–51). complex oligosaccharides are of special importance because they bear a negative charge. Whether a given oligosaccharide remains high-mannose or is processed depends largely on its position in the protein. If the oligosaccharide is accessible to the processing enzymes in the Golgi apparatus, it is likely to be converted to a complex form; if it is inaccessible because its sugars are tightly held to the protein’s surface, it is likely to remain in a high-mannose form. The processing that generates complex oligosaccharide chains follows the highly ordered pathway shown in Figure 13–31.


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Cell_Biology_Alberts. Beyond these commonalities in oligosaccharide processing that are shared among most cells, the products of the carbohydrate modifications carried out in the Golgi apparatus are highly complex and have given rise to a new field of study called glycobiology. The human genome, for example, encodes hundreds of different Golgi glycosyl transferases and many glycosidases, which are expressed differently from one cell type to another, resulting in a variety of glycosylated forms of a given protein or lipid in different cell types and at varying stages of differentiation, depending on the spectrum of enzymes expressed by the cell. The complexity of modifications is not limited to N-linked oligosaccharides but also occurs on O-linked sugars, as we discuss next. proteoglycans are assembled in the golgi apparatus


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Cell_Biology_Alberts. proteoglycans are assembled in the golgi apparatus In addition to the N-linked oligosaccharide alterations made to proteins as they pass through the Golgi cisternae en route from the ER to their final destinations, many proteins are also modified in the Golgi apparatus in other ways. Some proteins have sugars added to the hydroxyl groups of selected serines or threonines, H H NHCOCH3 remainder of H NHCOCH3 oligosaccharide side chain remainder of oligosaccharide side chain or, in some cases—such as collagens—to hydroxylated proline and lysine side chains. This O-linked glycosylation (Figure 13–32), like the extension of N-linked oligosaccharide chains, is catalyzed by a series of glycosyl transferase enzymes that use the sugar nucleotides in the lumen of the Golgi apparatus to add sugars to a protein one at a time. Usually, N-acetylgalactosamine is added first, followed by a variable number of additional sugars, ranging from just a few to 10 or more.


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Cell_Biology_Alberts. The Golgi apparatus confers the heaviest O-linked glycosylation of all on mucins, the glycoproteins in mucus secretions, and on proteoglycan core proteins, which it modifies to produce proteoglycans. As discussed in Chapter 19, this process involves the polymerization of one or more glycosaminoglycan chains (long, unbranched polymers composed of repeating disaccharide units; see Figure 19–35) onto serines on a core protein. Many proteoglycans are secreted and become components of the extracellular matrix, while others remain anchored to the extracellular face of the plasma membrane. Still others form a major component of slimy materials, such as the mucus that is secreted to form a protective coating on the surface of many epithelia.


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Cell_Biology_Alberts. The sugars incorporated into glycosaminoglycans are heavily sulfated in the Golgi apparatus immediately after these polymers are made, thus adding a significant portion of their characteristically large negative charge. Some tyrosines in proteins also become sulfated shortly before they exit from the Golgi apparatus. In both cases, the sulfation depends on the sulfate donor 3ʹ-phosphoadenosine-5ʹ-phosphosulfate (PAPS) (Figure 13–33), which is transported from the cytosol into the lumen of the trans Golgi network. what Is the purpose of glycosylation?


Cell_Biology_Alberts

Cell_Biology_Alberts. what Is the purpose of glycosylation? There is an important difference between the construction of an oligosaccharide and the synthesis of other macromolecules such as DNA, RNA, and protein. Whereas nucleic acids and proteins are copied from a template in a repeated series of identical steps using the same enzyme or set of enzymes, complex carbohydrates require a different enzyme at each step, each product being recognized as the exclusive substrate for the next enzyme in the series. The vast abundance of glycoproteins and the complicated pathways that have evolved to synthesize them emphasize that the oligosaccharides on glycoproteins and glycosphingolipids have very important functions.


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Cell_Biology_Alberts. N-linked glycosylation, for example, is prevalent in all eukaryotes, including yeasts. N-linked oligosaccharides also occur in a very similar form in archaeal cell wall proteins, suggesting that the whole machinery required for their synthesis is evolutionarily ancient. N-linked glycosylation promotes protein folding in two ways. First, it has a direct role in making folding intermediates more soluble, thereby preventing their aggregation. Second, the sequential modifications of the N-linked oligosaccharide establish a “glyco-code” that marks the progression of Figure 13–32 Nand O-linked glycosylation. In each case, only the single sugar group that is directly attached to the protein chain is shown. Figure 13–33 The structure of PAPS. protein folding and mediates the binding of the protein to chaperones (discussed in Chapter 12) and lectins—for example, in guiding ER-to-Golgi transport. As we discuss later, lectins also participate in protein sorting in the trans Golgi network.


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Cell_Biology_Alberts. Because chains of sugars have limited flexibility, even a small N-linked oligosaccharide protruding from the surface of a glycoprotein (Figure 13–34) can limit the approach of other macromolecules to the protein surface. In this way, for example, the presence of oligosaccharides tends to make a glycoprotein more resistant to digestion by proteolytic enzymes. It may be that the oligosaccharides on cell-surface proteins originally provided an ancestral cell with a protective coat; compared to the rigid bacterial cell wall, such a sugar coat has the advantage that it leaves the cell with the freedom to change shape and move.


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Cell_Biology_Alberts. The sugar chains have since become modified to serve other purposes as well. The mucus coat of lung and intestinal cells, for example, protects against many pathogens. The recognition of sugar chains by lectins in the extracellular space is important in many developmental processes and in cell–cell recognition: selectins, for example, are transmembrane lectins that function in cell–cell adhesion during blood cell migration, as discussed in Chapter 19. The presence of oligosaccharides may modify a protein’s antigenic and functional properties, making glycosylation an important factor in the production of proteins for pharmaceutical purposes.


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Cell_Biology_Alberts. Glycosylation can also have important regulatory roles. Signaling through the cell-surface signaling receptor Notch, for example, is an important factor in determining the cell’s fate in development (discussed in Chapter 21). Notch is a transmembrane protein that is O-glycosylated by addition of a single fucose to some serines, threonines, and hydroxylysines. Some cell types express an additional glycosyl transferase that adds an N-acetylglucosamine to each of these fucoses in the Golgi apparatus. This addition changes the specificity of Notch for the cell-surface signal proteins that activate it. Transport Through the golgi apparatus May occur by cisternal Maturation


Cell_Biology_Alberts

Cell_Biology_Alberts. It is still uncertain how the Golgi apparatus achieves and maintains its polarized structure and how molecules move from one cisterna to another, and it is likely that more than one mechanism is involved in each case. One hypothesis, called the cisternal maturation model, views the Golgi cisternae as dynamic structures that mature from early to late by acquiring and then losing specific Golgi-resident proteins. According to this view, new cis cisternae continually form as vesicular tubular clusters arrive from the ER and progressively mature to become a medial cisterna and then a trans cisterna (Figure 13–35A). A cisterna therefore moves through the Golgi stack with cargo in its lumen. Retrograde transport of the Golgi enzymes by budding COPI-coated vesicles explains their characteristic distribution. As we discuss later, when a cisterna finally moves forward to become part of the trans Golgi network, various types of coated vesicles bud off it until this network disappears, to be


Cell_Biology_Alberts

Cell_Biology_Alberts. distribution. As we discuss later, when a cisterna finally moves forward to become part of the trans Golgi network, various types of coated vesicles bud off it until this network disappears, to be replaced by a maturing cisterna just behind. At the same time, other transport vesicles are continually retrieving membrane from post-Golgi compartments and returning it to the trans Golgi network.


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Cell_Biology_Alberts. The cisternal maturation model is supported by studies using Golgi enzymes from different cisternae that were fluorescently labeled with different colors. Such studies performed in yeast cells where Golgi cisternae are not stacked reveal that individual Golgi cisternae change their color, thereby demonstrating that they change their complement of resident enzymes as they mature, even though they are not stacked. In further support of the model, electron microscopic observations found that large structures such as procollagen rods in fibroblasts and scales in certain algae move progressively through the Golgi stack. An alternative view holds that Golgi cisternae are long-lived structures that retain their characteristic set of Golgi-resident proteins firmly in place, and cargo proteins are transported from one cisterna to the next by transport vesicles (Figure 13–35B). According to this vesicle transport model, retrograde flow of vesicles


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Cell_Biology_Alberts. Figure 13–34 The three-dimensional structure of a small N-linked oligosaccharide. The structure was determined by x-ray crystallographic analysis of a glycoprotein. This oligosaccharide contains only 6 sugars, whereas there are 14 sugars in the n-linked oligosaccharide that is initially transferred to proteins in the er (see figure 12–47). (a) a backbone model showing all atoms except hydrogens; only the asparagine of the protein is shown. (b) a space-filling model, with the asparagine and sugars indicated using the same color scheme as in (a). (b, courtesy of richard feldmann.) Figure 13–35 Two possible models explaining the organization of the Golgi apparatus and how proteins move through it. It is likely that the transport through the golgi apparatus in the forward direction (red arrows) involves elements of both models.


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Cell_Biology_Alberts. (a) according to the cisternal maturation model, each golgi cisterna matures as it migrates outward through the stack. at each stage, the golgi resident proteins that are carried forward in a maturing cisterna are moved backward to an earlier compartment in copI-coated vesicles. when a newly formed cisterna moves to a medial position, for example, “leftover” cis golgi enzymes would be extracted and transported retrogradely to a new cis cisterna behind. likewise, the medial enzymes would be received by retrograde transport from the cisternae just ahead. In this way, a cis cisterna would mature to a medial and then trans cisterna as it moves outward. (b) In the vesicle transport model, golgi cisternae are static compartments, which contain a characteristic complement of resident enzymes. The passing of molecules from cis to trans through the golgi is accomplished by forward-moving transport vesicles, which bud from one cisterna and fuse with the next in a cis-to-trans direction.


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Cell_Biology_Alberts. retrieves escaped ER and Golgi proteins and returns them to upstream compartments. Directional flow could be achieved because forward-moving cargo molecules are selectively packaged into forward-moving vesicles. Although both forwardand backward-moving vesicles would likely be COPI-coated, the coats may contain different adaptor proteins that confer selectivity on the packaging of cargo molecules. Alternatively, transport vesicles shuttling between Golgi cisternae might not be directional at all, transporting cargo randomly back and forth; directional flow would then occur because of the continual input to the cis cisterna and output from the trans cisterna.


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Cell_Biology_Alberts. The vesicle transport model is supported by experiments that show that cargo molecules are present in small COPI-coated vesicles and that these vesicles can deliver them to Golgi cisternae over large distances. In addition, when experimentally aggregated membrane proteins are introduced into Golgi cisternae, they can be observed staying in place, while soluble cargo, even if present as large aggregates, traverses the Golgi at normal rates.


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Cell_Biology_Alberts. It is likely that aspects of both models are true. A stable core of long-lasting cisternae might exist in the center of each Golgi cisterna, while regions at the rim may undergo continuous maturation, perhaps utilizing Rab cascades that change their identity. As matured pieces of the cisternae are formed, they might break off and fuse with downstream cisternae by homotypic fusion mechanisms, taking large cargo molecules with them. In addition, small COPI-coated vesicles might transport small cargo in the forward direction and retrieve escaped Golgi enzymes and return them to their appropriate upstream cisternae. Golgi golgi Matrix proteins help organize the stack


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Cell_Biology_Alberts. The unique architecture of the Golgi apparatus depends on both the microtubule cytoskeleton, as already mentioned, and cytoplasmic Golgi matrix proteins, which form a scaffold between adjacent cisternae and give the Golgi stack its structural integrity. Some of the matrix proteins, called golgins, form long tethers composed of stiff coiled-coil domains with interspersed hinge regions. Golgins form a forest Figure 13–36 A model of golgin function. of tentacles that can extend 100–400 nm from the surface of the Golgi stack. They filamentous golgins anchored to golgi are thought to help retain Golgi transport vesicles close to the organelle through membranes capture transport vesicles interactions with Rab proteins (Figure 13–36). When the cell prepares to divide, by binding to rab proteins on the vesicle mitotic protein kinases phosphorylate the Golgi matrix proteins, causing the Golgi surface.


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Cell_Biology_Alberts. apparatus to fragment and disperse throughout the cytosol. The Golgi fragments are then distributed evenly to the two daughter cells, where the matrix proteins are dephosphorylated, leading to the reassembly of the Golgi stack. Similarly, during apoptosis, proteolytic cleavage of golgins by caspases ensues (discussed in Chapter 18), fragments the Golgi apparatus as the cell self-destructs. Correctly folded and assembled proteins in the ER are packaged into COPII-coated transport vesicles that pinch off from the ER membrane. Shortly thereafter, the vesicles shed their coat and fuse with one another to form vesicular tubular clusters. In animal cells, the clusters then move on microtubule tracks to the Golgi apparatus, where they fuse with one another to form the cis Golgi network. Any resident ER proteins that escape from the ER are returned there from the vesicular tubular clusters and Golgi apparatus by retrograde transport in COPI-coated vesicles.


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Cell_Biology_Alberts. The Golgi apparatus, unlike the ER, contains many sugar nucleotides, which glycosyl transferase enzymes use to glycosylate lipid and protein molecules as they pass through the Golgi apparatus. The mannoses on the N-linked oligosaccharides that are added to proteins in the ER are often initially removed, and further sugars are added. Moreover, the Golgi apparatus is the site where O-linked glycosylation occurs and where glycosaminoglycan chains are added to core proteins to form proteoglycans. Sulfation of the sugars in proteoglycans and of selected tyrosines on proteins also occurs in a late Golgi compartment.


Cell_Biology_Alberts

Cell_Biology_Alberts. The Golgi apparatus modifies the many proteins and lipids that it receives from the ER and then distributes them to the plasma membrane, lysosomes, and secretory vesicles. The Golgi apparatus is a polarized organelle, consisting of one or more stacks of disc-shaped cisternae. Each stack is organized as a series of at least three functionally distinct compartments, termed cis, medial, and trans cisternae. The cis and trans cisternae are each connected to special sorting stations, called the cis Golgi network and the trans Golgi network, respectively. Proteins and lipids move through the Golgi stack in the cis-to-trans direction. This movement may occur by vesicle transport, by progressive maturation of the cis cisternae as they migrate continuously through the stack, or, most likely, by a combination of these two mechanisms. Continual retrograde vesicle transport from upstream to more downstream cisternae is thought to keep the enzymes concentrated in the cisternae where they are


Cell_Biology_Alberts

Cell_Biology_Alberts. by a combination of these two mechanisms. Continual retrograde vesicle transport from upstream to more downstream cisternae is thought to keep the enzymes concentrated in the cisternae where they are needed. The finished new proteins end up in the trans Golgi network, which packages them in transport vesicles and dispatches them to their specific destinations in the cell.


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Cell_Biology_Alberts. TransporT froM The Trans golgI neTwork To lysosoMes The trans Golgi network sorts all of the proteins that pass through the Golgi apparatus (except those that are retained there as permanent residents) according to their final destination. The sorting mechanism is especially well understood for those proteins destined for the lumen of lysosomes, and in this section we consider this selective transport process. We begin with a brief account of lysosome structure and function. lysosomes are the principal sites of Intracellular digestion


Cell_Biology_Alberts

Cell_Biology_Alberts. lysosomes are the principal sites of Intracellular digestion Lysosomes are membrane-enclosed organelles filled with soluble hydrolytic enzymes that digest macromolecules. Lysosomes contain about 40 types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases. All are acid hydrolases; that is, hydro-lases that work best at acidic pH. For optimal activity, they need to be activated by proteolytic cleavage, which also requires an acid environment. The lysosome provides this acidity, maintaining an interior pH of about 4.5–5.0. By this arrange- ment, the contents of the cytosol are doubly protected against attack by the cell’s own digestive system: the membrane of the lysosome keeps the digestive enzymes out of the cytosol, but, even if they leak out, they can do little damage at the cytosolic pH of about 7.2.


Cell_Biology_Alberts

Cell_Biology_Alberts. Like all other membrane-enclosed organelles, the lysosome not only contains a unique collection of enzymes, but also has a unique surrounding membrane. Most of the lysosome membrane proteins, for example, are highly glycosylated, which helps to protect them from the lysosome proteases in the lumen. Transport proteins in the lysosome membrane carry the final products of the digestion of macromolecules—such as amino acids, sugars, and nucleotides—to the cytosol, where the cell can either reuse or excrete them.


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Cell_Biology_Alberts. A vacuolar H+ ATPase in the lysosome membrane uses the energy of ATP hydrolysis to pump H+ into the lysosome, thereby maintaining the lumen at its acidic pH (Figure 13–37). The lysosome H+ pump belongs to the family of V-type ATPases and has a similar architecture to the mitochondrial and chloroplast ATP synthases (F-type ATPases), which convert the energy stored in H+ gradients into ATP (see Figure 11–12). By contrast to these enzymes, however, the vacuolar H+ ATPase exclusively works in reverse, pumping H+ into the organelle. Similar or identical V-type ATPases acidify all endocytic and exocytic organelles, including lysosomes, endosomes, some compartments of the Golgi apparatus, and many transport and secretory vesicles. In addition to providing a low-pH environment that is suitable for reactions occurring in the organelle lumen, the H+ gradient provides a source of energy that drives the transport of small metabolites across the organelle membrane. lysosomes are heterogeneous


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Cell_Biology_Alberts. lysosomes are heterogeneous Lysosomes are found in all eukaryotic cells. They were initially discovered by the biochemical fractionation of cell extracts; only later were they seen clearly in the electron microscope. Although extraordinarily diverse in shape and size, staining them with specific antibodies shows they are members of a single family of organelles. They can also be identified by histochemical techniques that reveal which organelles contain acid hydrolase (Figure 13–38).


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Cell_Biology_Alberts. The heterogeneous morphology of lysosomes contrasts with the relatively uniform structures of many other cell organelles. The diversity reflects the wide variety of digestive functions that acid hydrolases mediate, including the breakdown of intraand extracellular debris, the destruction of phagocytosed microorganisms, and the production of nutrients for the cell. Their morphological diversity, however, also reflects the way lysosomes form. Late endosomes containing material received from both the plasma membrane by endocytosis and newly synthesized lysosomal hydrolases fuse with preexisting lysosomes to form structures that are sometimes referred to as endolysosomes, which then fuse with one another (Figure 13–39). When the majority of the endocytosed material within an endolysosome has been digested so that only resistant or slowly digestible residues remain, these organelles become “classical” lysosomes. These are relatively dense, round, and small, but they can enter the cycle


Cell_Biology_Alberts

Cell_Biology_Alberts. been digested so that only resistant or slowly digestible residues remain, these organelles become “classical” lysosomes. These are relatively dense, round, and small, but they can enter the cycle again by fusing with late endosomes or endolysosomes. Thus, there is no real distinction between endolysosomes and lysosomes: they are the same except that they are in different stages of a maturation cycle. For this reason, lysosomes are sometimes viewed as a heterogeneous collection of distinct organelles, the common feature of which is a high content of


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Cell_Biology_Alberts. Figure 13–38 Histochemical visualization of lysosomes. These electron micrographs show two sections of a cell stained to reveal the location of acid phosphatase, a marker enzyme for lysosomes. The larger membrane-enclosed organelles, containing dense precipitates of lead phosphate, are lysosomes. Their diverse morphology reflects variations in the amount and nature of the material they are digesting. The precipitates are produced when tissue fixed with glutaraldehyde (to fix the enzyme in place) is incubated with a phosphatase substrate in the presence of lead ions. red arrows in the top panel indicate two small vesicles thought to be carrying acid hydrolases from the golgi apparatus. (courtesy of daniel s. friend.) 0.2–0.5 µm pH~7.2 pH~5.0 ACID HYDROLASES: nucleases proteases glycosidases lipases phosphatases sulfatases phospholipases CYTOSOL + H+ pump ATPADPPi H+ Figure 13–37 Lysosomes. The acid hydrolases are hydrolytic enzymes that are active under acidic conditions. an h+ aTpase