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Roadmap: Proteins are translated into the ER, which are translocated into the lumen. They are then sorted into vesicles which leave the ER for the Golgi, and then out to the plasma membrane. From the plasma membrane they might go back to the endosomes, and from the endosomes they might head back to the Golgi. Vesicle Transport There are multiple donor and target membranes in the cell, so targeting information is needed to determine specificity of vesicle targeting. There are two mechanisms to determine this specificity: the Rab GTPase tethering mechanisms, and the SNARE fusion machinery. Cytoskeleton is the tracks of transport for vesicles Cytoskeleton is made up of protein filaments (actin) and microtubules (α and β tubulin), and it runs through the cytosol to provide structure to cells. Actin filaments are shorter and often clustered at the PM; they are highly crosslinked into dynamic networks. Microtubules are longer, and are usually organized around centrosome near nucleus, though they can extend into the periphery of the cells. The two are connected to each other and anchored to PM and organelles. Transport of vesicles is mediated by motor proteins, which move along the cytoskeleton. There are two types of motor proteins: myosins move on actin filaments, while dyneins and kinesins move on microtubules. These motors transport vesicles and other components along the cytoskeleton, but despite being ATP-dependent (dynein is actually an AAA protein), they do not specify target. Rab proteins Rab proteins are in a large family of monomeric GTPases (same family as the Sar1 and Arf, which are responsible for vesicle formation) and come in different sets, each uniquely associated with an organelle and vesicle type in the secretory pathway. The unique combination gives the targeting specificity of the vesicle. They have two geranylgeranyl (lipid) groups at the C-termini (to insert into the membrane); however, Rabs can be removed from the membrane by specific protein interactions. In general, the GTP-bound state is the active state (GDP-bound is off), which interacts with various effector proteins and membranes. The Rab Cycle starts with GDP-bound, inactive Rab 1. The inactive Rab-GDP is bound by GDI (GDP dissociation inhibitor) a. GDI covers the geranylgeranyls, which makes the inactive Rab-GDP soluble and not membrane-associated 2. To turn on Rab, GDF (GDP Dissociation Factor) and GEF (GTP Exchange Factor) on the membrane produce Rab-GTP with exposed geranylgeranyl lipids that can become anchored to the membrane 3. The activated Rab-GTP works through effector proteins on the vesicle and acceptor membrane, the interactions of which determine target specificity 4. After targeting has been accomplished GAP (GTPase activating protein) induces Rab to hydrolyze GTP, and makes it go back to GDP form 5. Rab Escort Proteins and Geranylgeranyl-transferase prepare newly-synthesized Rabs Rab Functions in sorting and directing Rab assists in sorting of cargo receptors into vesicles on donor membranes during budding. (Rab-GDP’s GDP is taken off by GDF, at the same time that the GEF replaces the empty GDP slot with the GTP; interactions between Rab and cargo receptors may help gather cargo into vesicles). Also, Rab might regulate PI-phosphates by recruiting PI-kinases or PI-phosphatases to control type of PI-lipids on a membrane. A little evidence that Rab might connect the vesicles to the cytoskeletal models, though it might not be universal. A more universal function is that Rabs are able to initiate targeting of vesicle by connecting the Rab effector proteins via tethering. Tethering involves the approaching of the vesicle to the acceptor membrane, and the Rabs on the vesicle and on the acceptor will activate long tethering proteins to help bring the vesicle to the right acceptor. Most Rab effectors, in fact, are tethers—long proteins that connect the vesicle and acceptor membranes. Rab/Tether interactions are the primary determinant of vesicle targeting specificity. Also, the Rab-tether interactions might also prepare the vesicle fusion machinery (SNARE) on the vesicle and acceptor membranes (they don’t mediate the fusion, but they help SNAREs mediate the fusion). More about Tethers: multitasking Coiled-coils, and single-tasking Multisubunits Multisubunit tethers direct vesicle targeting to the major secretory compartment membranes. For example, the exocyst (first identified in yeast, but also has parallels in humans) is a multisubunit tether for vesicle fusion at PM and endosomes. Some components of the exocyst are carried on vesicles, while others on acceptor membranes. The two parts come together to form large octameric complexes attached to Rabs. A similar protein to Rab is Rho, which is a regulator on the PM and could form the octameric complex tethers with Rabs. Another type of tether is the Coiled-coil tethers, which are special dimeric tethers with long coiled-coil structures (pairs of α-helices wrapped around each other) that connect vesicles to Golgi membranes. The Coiled-coil tethers are also responsible for the shape of the Golgi stack in its layered form (second function). On the other hand, they are not related to the multisubunit tethers, and attach to membranes via anchors or interactions with Rab and Arf. Some Rab effectors (other than tethers) have GEF or PI kinase/phosphatase activities. This means that when an activated Rab turn on an effector, the effector is actually able to turn on another Rab (through GEF activity) within proximity to it. This positive feedback loop (Rab-GTP producing more Rab-GTP in a localized spot in the membrane) contributes to the clustering of tethers in the site of fusion. Also, the PI-phosphates phosphorylates other PIs, which provide additional binding sites for Rab effectors such as tethers. Targeting and Fusion: SNAREs also determine specificity SNARE proteins are a family of related membrane proteins. Specific v-SNAREs on vesicles recognize specific partner t-SNAREs on target membranes. The SNAREs function after tethering and are the second determinant of targeting specificity. Formation of SNARE complexes between the vesicles and acceptor membranes mediates fusion. V-Snares are monomers with single TM domains. T-SNAREs are trimers with one TM domain and two peripheral subunits. The correct set of v- and t-SNAREs form a stable tetramer, 4 tightly wound α-helices in a bundle, the formation energy of which drives fusion. Fusion Mechanism SNARE complex structure (coiled-coil interactions) resembles certain viral coat proteins. For viruses, they fuse their membranes with host cell PM during infection. The mechanism of fusion is mediated by the coat proteins—which bind to the host membrane, then change conformation in helical domains to cause fusion. This gave rise to SNARE hypothesis: vesicle fusion was triggered by conformational changes in SNAREs, the same way that viral coat proteins change conformation in helical domains to fuse. The mechanism of SNARE fusion is as follows: 1. V-SNARE monomer, before fusion, is not stably folded. The majority of its cytosolic domain is flopping around. However, it folds into helical bundles when brought together with t-SNAREs (which are also originally unstable, but since they have two peripheral subunits, they are somewhat more stable than vSNAREs) 2. The base of the v-SNARE eventually becomes close with the base of the tSNARE. Thus, the folding process pulls the membranes close together and generates physical strain (like a spring). a. This is NOT dependent on ATP/GTP! 3. The membranes are held together in such a way that water is excluded from the area a. This exclusion allows hydrophobic contacts between lipids of membranes i. Outer leaflet (outer layer of lipids) joining = hemifusion; this is an unstable intermediate ii. Inner leaflet fusion (inner layer of lipids) joining = fusion; this is the stable final product, which relieves strain from the SNARE complex SNARE Dissociation After fusion, the SNARE complex has had all of its strain relieved, and is consequently stable and inactive. However, the v- and t-SNAREs are still hooked up together on the acceptor membrane; what separates them? NSF is an AAA-family ATPase protein that separates and recycles v- and t-SNAREs on the acceptor protein, which is essential for continuation of vesicle traffic. (Note the theme of AAA: their favorite pastimes seems to be pulling things apart—pulling apart ER lumen proteins, proteasomal proteins, and now pulling apart SNAREs). With accessory proteins and ATP, it rips the v-t complex apart. The t-SNAREs are active again, and the v-SNAREs are recycled back to their donor membranes by other vesicles. Homotypic Fusion: when donor and target membranes are the same Examples of homotypic fusion are when COP-II vesicles make the VTC (vesiculartubular cluster), or the re-formation of organelles after cell division. In these cases, both membranes have identical v- and t-SNAREs. Therefore, in these cases, SNAREs mut be separated by NSF to allow fusion. (So the last step becomes the first step). 1. t- and v-SNAREs are separated on vesicles by NSF 2. NSF dissociates and the two vesicles fuse by their respective t-v-SNAREs in homotypic fusion 3. Now there are two sets of v-t-SNARE complexes on one vesicle