Bi1_2011_PS1_AdditionalReading

Bi1_2011_PS1_AdditionalReading - 766 Chapter 13...

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Unformatted text preview: 766 Chapter 13: Intracellular Vesicular Traffic Summary Directed and selective transport of particular membrane components from one mem- brane-enclosed compartment of a eucaryotic cell to another maintains the differences between those compartments. Transport vesicles, which can be spherical, tubular, 0r 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. 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 COPI- and COPII-coated vesicles, which mediate transport between Golgi cisternae and between the ER and the Golgi apparatus, respectively. In clathrin- coated vesicles, adaptor proteins link the clathrin to the vesicle membrane and also trap specific cargo molecules for packaging into the vesicle. The coat is shed rapidly after budding, enabling the vesicle to fuse with its appropriate target membrane. Local synthesis of phosphoinositides creates binding sites that trigger coat assem- bly and vesicle budding. In addition, monomeric GTPases help regulate various steps in vesicular transport, including both vesicle budding and docking. The coat-recruitment GTPases, including Sarl 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 transport vesicles and target membranes. The assembly and disassembly of Rab proteins and their eflectors in specialized membrane domains are dynamically controlled by GTP binding and hydrolysis. Active Rab proteins recruit Rab eflectors, such as motor proteins, which transport vesicles on actin filaments or microtubules, and filamentous tethering proteins, which help ensure that the vesicles deliver their contents only to the appropriate membrane-enclosed compartment. 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 apposi- tion so that their lipid bilayers can fuse. 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 biosynthetic—secretory pathway. During their sub- ENDOPLASM‘F RETICULUM sequent transport, from the ER to the Golgi apparatus and from the Golgi appa- ratus to the cell surface and elsewhere, these proteins are successively modified _ as they pass through a series of compartments. Transfer from one compartment l _ to the next involves a delicate balance between forward and backward (retrieval) 'l ll- transport pathways. Some transport vesicles select cargo molecules and move ; them to the next compartment in the pathway, while others retrieve escaped ' ' l I SECRUORY LATE ENDOSOME VESICLES proteins and return them to a previous compartment where they normally func- -— ‘ tion. Thus, the pathway from the ER to the cell surface consists of many sorting _ ' steps, which continuously select membrane and soluble lumenal proteins for “flown—NE packaging and transport—in vesicles or organelle fragments that bud from the ER and Golgi apparatus. In this section we focus mainly on the Golgi apparatus (also called the Golgi EARLY Enposomg ' I 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 a —.\ — .— in the Golgi apparatus, including the pectin and hemicellulose of the cell wall in : CELL EXTERTOR 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 pro- teins 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. TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS Proteins Leave the ER in COPII-Coated Transport Vesicles To initiate their journey along the biosynthetic—secretory pathway, proteins that have entered the ER and are destined for the Golgi apparatus or beyond are first packaged into small 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. Originally, it was thought that all proteins that are not tethered in the ER enter transport vesicles by default. It is now clear, however, that entry into vesi— cles that leave the ER is usually a selective process. Many membrane proteins are actively recruited into such vesicles, where they become concentrated. It is thought that these cargo proteins display exit (transport) signals on their cytoso- lic surface that components of the COPII coat recognize (Figure 13—20); these coat 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, which in turn bind through exit signals in their cytoplasmic tails to components of the COPII coat. At a lower rate, proteins without exit signals can also enter transport vesicles, so that even proteins that normally function in the ER (so-called ER resident proteins) slowly leak out of the ER and are delivered to the Golgi apparatus. Similarly, secretory proteins that are made in high con- centrations may leave the ER without the help of exit signals or cargo receptors. 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. The ERGIC53 lectin, for example, binds to mannose and is thought to recognize this sugar on two secreted blood-clotting factors (FactorV and Factor VIII), thereby packaging the proteins into transport vesicles in the ER. ERGIC53’s role in protein transport was identified because humans who lack it owing to an inherited mutation have lowered serum levels of FactorsV and VIII, and they therefore bleed excessively. Only Proteins That Are Properly Folded and Assembled Can Leave the ER To exit from the ER, proteins must be properly folded and, if they are subunits of multimeric protein complexes, they may need to be completely assembled. Those that are misfolded or incompletely assembled remain in the ER, where they are bound to chaperone proteins (discussed in Chapter 6), such as BiP or calnexiri. The chaperones may cover up the exit signals or somehow anchor the proteins in forming ER transport vesicle -Sar1-GTP - —- subunits of exit signal on COPII coat membrane—bound cargo pretem \ exit signal on cargo receptor CYTOSOL ' exit signal on cargo receptor—— resident ER ‘ protein ER LUMEN ‘ exit signal on soluble cargo M protein chaperone proteins bound to unfolded or misfolded proteins 767 Figure 13—20 The recruitment of cargo molecules into ER transport vesicles. By binding directly or indirectly to the COPII coat, membrane and soluble cargo proteins, respectively, become concentrated in the transport vesicles as they leave the ER. Membrane proteins are packaged into budding transport vesicles through interactions of exit signals on their cytosolic tails with the COPII coat. Some of the membrane proteins that the coat traps function as cargo receptors, binding soluble proteins in the lumen and helping to package them into vesicles. A typical SO—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 retained in the ER compartment. 768 Chapter 13: Intracellular Vesicular Traffic budding transport vesic III! ‘\ antibody heavy chain antibody light chain ramr/ RETAINED IN ER CYTOSOL SECRETED the ER (Figure 13—21). 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 mis- assembled proteins that could potentially interfere with the functions of normal proteins. There is a surprising amount of corrective action. More than 90% of the newly synthesized subunits of the T cell receptor (discussed in Chapter 25) 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 many protein molecules to produce a select few that fold, assemble, and function properly. The process of continual degradation of a portion of ER proteins also pro- vides an early warning system to alert the immune system when a virus infects cells. Using specialized ABC-type transporters, the ER imports peptide frag— ments of viral proteins produced by proteases in the proteasome. The foreign peptides are loaded onto class I MHC proteins in the ER lumen and then trans- ported to the cell surface. T lymphocytes then recognize the peptides as non-self antigens and kill the infected cells (discussed in Chapter 25). 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 C1‘ transport. Although the mutant protein would function perfectly normally if it reached the plasma membrane, it remains in the ER. This devastating disease thus results not because the muta- tion 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 After transport vesicles have budded from ER exit sites and have shed their coat, they begin to fuse with one another. This 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 sym- metrical, with both membranes contributing v—SNAREs and t—SNAREs (Figure 13—22). The structures formed when ER-derived vesicles fuse with one another are called vesicular tubular clusters, because they have a convoluted appearance in Figure 13-21 Retention of incompletely assembled antibody molecules in the ER. Antibodies are made up of two heavy and two light chains (discussed in Chapter 25), which assemble in the ER. The chaperone BiP is thought to bind to all incompletely assembled antibody molecules and to cover up an exit signal. Thus, only completely assembled antibodies leave the ER and are secreted. TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS homotypic membrane fusion —--I- Q. 0 mi fl} 1’ STEP 2 STEP 3 the electron microscope (Figure 13—23A). These clusters constitute a new com- partment 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 are relatively short-lived because they move quickly along microtubules to the Golgi apparatus, with which they fuse to deliver their contents (Figure 13—23B). 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. They carry back to the ER resident proteins that have escaped, as well as proteins such as cargo receptors that participated in the ER budding reaction and are being returned. This retrieval process demon- strates the exquisite control mechanisms that regulate coat assembly reactions. The COPI coat assembly begins only seconds after the COPII coats have been shed. It remains a mystery how this switch in coat assembly is controlled. The retrieval (or retrograde) transport continues as the vesicular tubular clusters move towards the Golgi apparatus. Thus, the clusters continuously mature, gradually changing their composition as selected proteins are returned to the ER. A similar retrieval process 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 sig- nals that bind directly to COPI coats and are thus packaged into COPIAcoated vesicular tubular cluster COP“ (oat motor protein — '- ER (3) \ a veilcular ,2 tubular 0 cluster ._ 0 4—4 0 / retrieval transport 769 Figure 13—22 Homotypic membrane fusion. <AAAA> In step 1, NSF pries apart identical pairs of v—SNAREs and t—SNAREs in both membranes (see Figure 13—18). 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 called a vesicular tubular cluster. Subsequently, the compartment grows by further homotypic fusion with vesicles from the same kind of membrane, displaying matching SNAREs. Homotypic fusion is not restricted to the formation of vesicular tubular clusters; in a similar process, endosomes fuse to generate larger endosomes. Rab proteins help regulate the extent of homotypic fusion and hence the size of the compartments in a cell (not shown). Figure 13—23 Vesicular tubular clusters. (A) An electron micrograph section of vesicular tubular clusters forming from the ER membrane. 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 to the Golgi apparatus. COPI coats mediate the budding of vesicles that return to the ER from these clusters. As indicated, the coats quickly disassemble after the vesicles have formed. (A, courtesy ofWilliam Balch.) microtubule __... y as Golgi network com _ coat 770 Chapter 13: Intracellular Vesicular Traffic vesicular tubular cluster or Gulgl apparatus soluble ER -. . . protein resrdent protein secretory COPII F D RWA R D PATHWAY ‘ KDEL empty - KDEL receptor J o 3' KDEL protein _ l receptor . a # RETRIEVAL -‘ com 3!. mat soluble PATHWAY (99 (A) ERresident @ v___ _ protein _' u " L—__i ER (B) 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 KIOQC sequence, based on the single-letter amino acid code. Soluble ER resident proteins, such as BiP, also contain a short 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. Unlike the retrieval signals on ER membrane proteins, which can interact directly with the COPI coat, soluble ER resident proteins must bind to special- ized receptor proteins such as the KDEL receptor—a multipass transmembrane protein that binds to the KDEL sequence and packages any protein displaying it into COPl-coated retrograde transport vesicles (Figure 13—24). 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 com- partments. The receptor must have a high affinity for the KDEL sequence in vesicular tubular clusters and the Golgi apparatus, so as to capture escaped, sol- uble 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 resident proteins in the ER. How does the affinity of the KDEL receptor change depending on the com- partment in which it resides? The answer is not known, but it may be related to the different ionic conditions and pH in the different compartments, which are regulated by ion transporters in the compartment membrane. As we discuss later, pH-sensitive protein—protein interactions form the basis for many of the sorting steps in the cell. Most membrane proteins that function at the interface between the ER and Golgi apparatus, including v- and t-SNAREs and some cargo receptors, enter the retrieval pathway back to the ER. Whereas the recycling of some of these pro- teins is mediated by signals, as just described, for others no specific signal seems to be required. Thus, while retrieval signals increase the efficiency of the retrieval process, some proteins randomly enter budding vesicles destined for the ER and are returned to the ER at a slower rate. Many Golgi enzymes cycle constantly between the ER and the Golgi, but their rate of return to the ER is slow enough for most of the protein to be found in the Golgi apparatus. cis Golgi stack trans Golgi Golgi network network Figure 13~24 A model for the retrieval of soluble ER resident proteins. ER resident proteins that escape from the ER are returned by vesicular transport. (A) The KDEL receptor present in 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. Upon binding its ligands in this environment, 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 all 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. TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS Many Proteins Are Selectively Retained in the Compartments in Which They Function The KDEL retrieval pathway only partly explains how ER resident proteins are maintained in the ER. As expected, 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 anchors ER resident proteins and that only those proteins that escape this retention mechanism are captured and returned via the KDEL recep- tor. A suggested retention mechanism is that ER resident proteins bind to one another, thus forming complexes that are too big to enter transport vesicles effi- ciently. Because ER resident proteins are present in the ER at very high concen- trations (estimated to be millimolar), relatively low—affinity interactions would suffice to tie up most of the proteins in such complexes. Aggregation of proteins that function in the same compartment—called kin recognition—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 vesi- cles leaving the Golgi apparatus. The Golgi Apparatus Consists of an Ordered Series of Compartments Because of its large and regular structure, 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 usually consists of four to six cisternae (Figure 13—25), although some unicellular flagellates can have up to _ ‘ as FACE Golgi vesicle cis Golgi network (CGNJ cis cisterna medial cisterna trans Golgi network (TG N} (A) - _ nuclear envelope rough ER vesicular - tubular clusters ' cis Golgi network 771 Figure 13—25 The Golgi apparatus. (A) Three—dimensional reconstruction from electron micrographs ofthe Golgi apparatus in a secretory animal celI.The cis face of the Golgi stack is that closest to the ER. (B) A thin- section electron micrograph emphasizing the transitional zone between the ER and the Golgi apparatus in an animal cell. (C) An electron micrograph ofa Golgi apparatus in a plant cell (the green alga Chlamydomonas) seen in cross section. 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; C, courtesy of George Palade.) ...
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Bi1_2011_PS1_AdditionalReading - 766 Chapter 13...

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