Vesicles lie at the heart of intracellular transport of many proteins. Genetic approaches and cell-free systems have been used to elucidate the mechanisms of their formation and transport. The overall process is complex, and involves a variety of cytosolic and membrane proteins, GTP, ATP, and accessory factors. Budding, tethering, docking, and membrane fusion are key steps in the life cycles of vesicles, with the GTP-binding proteins, Sar1, ARF, and Rab acting as molecular switches. Sar1 is the protein involved in step 1 of formation of COPII vesicles, whereas ARF is involved in the formation of COPI and clathrin-coated vesicles. The functions of the various proteins involved in vesicle processing and the abbreviations used are shown in Table1.

Table1. Some Factors Involved in the Formation of Non–Clathrin-Coated Vesicles & Their Transport

There are common general steps in transport vesicle formation, vesicle targeting, and fusion with a target mem brane, irrespective of the membrane the vesicle forms from or its intracellular destination. The nature of the coat proteins, GTPases and targeting factors differ depending on where the vesicle forms from and its eventual destination. Anterograde transport from the ER to the Golgi involving COPII vesicles is the best studied example. The process can be considered to occur in eight steps (Figure 1). The basic concept is that each transport vesicle is loaded with specific cargo and also one or more v-SNAREproteins that direct targeting. Each tar get membrane bears one or more complementary t-SNARE proteins with which v-SNAREs interact, mediating SNARE protein-dependent vesicle–membrane fusion. In addition, Rab proteins also help direct the vesicles to specific mem branes and their tethering at a target membrane.

Fig1. Anterograde transport involving COPII vesicles. Step 1: Sar1 is activated when GDP exchanged for GTP and it becomes embedded in the ER membrane to form a focal point for bud formation. Step 2: Coat proteins bind to Sar1·GTP and cargo proteins become enclosed inside the vesicles. Step 3:The bud pinches off, formatting a complete coated vesicle. Vesicles move through cells along microtubules or actin filaments. Step 4:The vesicle is uncoated when bound GTP is hydrolyzed to GDP by Sar1. Step 5: Rab molecules are attached to vesicles after switching of Rab·GDP to Rab·GTP, a specific GEF. Rab effector proteins on target membranes bind to Rab·GTP, tethering the vesicles to the target membrane. Step 6: v-SNAREs pair with cognate t-SNAREs in the target membrane to form a four-helix bundle which docks the vesicles and initiates fusion. Step 7: When the v- and t-SNARES are closely aligned, the vesicle fuses with the membrane and the contents are released. GTP is then hydrolyzed to GDP, and the Rab·GDP molecules are released into the cytosol. An ATPase (NSF) and α-SNAP dissociate the four-helix bundle between the v- and t-SNARES so that they can be reused. Step 8: Rab and SNARE proteins are recycled for further rounds of vesicle fusion.

Step 1: Budding is initiated when the GTPase Sar1 is activated by binding of GTP in exchange for GDP via the action of the GEF Sec12p, switching it from a soluble to a membrane bound form via conformational change which exposes a hydrophobic tail. This enables it to become embedded in the ER membrane to form a focal point for vesicle assembly.

Step 2: For bud formation, various coat proteins bind to Sar1·GTP. In turn, membrane cargo proteins bind to the coat proteins eitherdirectlyor viaintermediary proteinsthat attach to coat proteins, and they then become enclosed in their appropriate vesicles. Soluble cargo proteins bind to receptor regions inside the vesicles. A number of signal sequences on cargo molecules have been identified. For example, KDEL sequences direct certain proteins in retrograde flow from the Golgi to the ER in COPI vesicles. Diacidic sequences (eg, Asp-X-Glu, X = any amino acid) and short hydrophobic sequences on membrane proteins destined for the Golgi membrane are involved in interactions with coat proteins of COPII vesicles. However, not all cargo molecules have a sorting signal. Some highly abundant secretory proteins travel to various cellular destinations in transport vesicles by bulk flow; that is, they enter into transport vesicles at the same concentration that they occur in the organelle. However, it appears that most proteins are actively sorted (concentrated) into transport vesicles and bulk flow is used by only a select group of cargo proteins. Additional coat proteins are assembled to complete bud formation. Coat proteins promote budding, contribute to the curvature of buds, and also help sort proteins.

Step 3: The bud pinches off, completing formation of the coated vesicle. The curvature of the ER membrane and protein–protein and protein-lipid interactions in the bud facilitate pinching off from ER exit sites. Vesicles move through cells along microtubules or along actin filaments.

 Step 4: Coat disassembly, or uncoating (involving dis sociation of Sar1 and the shell of coat proteins) follows hydrolysis of bound GTP to GDP by Sar1, promoted by a specific coat protein. Sar1 thus plays key roles in both assembly and dissociation of the coat proteins. Uncoating is necessary for fusion to occur.

Step 5: Vesicle targeting is achieved by attachment of Rab molecules to vesicles. Rabs are a family of Ras-like proteins required in several steps of intracellular protein transport and also in regulated secretion and endocytosis. They are small monomeric GTPases that attach to the cytosolic faces of budding vesicles in the GTP-bound state and are also present on acceptor membranes. Rab·GDP molecules in the cytosol are switched to Rab·GTP molecules by a specific GEF (see Table 49–9). Rab effector proteins on acceptor membranes bind to Rab·GTP, but not Rab GDP molecules, thus tethering the vesicles to the membranes.

 Step 6: v-SNAREs pair with cognate t-SNAREs in the target membrane to dock the vesicles and initiate fusion. Generally, one v-SNARE in the vesicle pairs with three t-SNAREs on the acceptor membrane to form a tightfour helix bundle. In synaptic vesicles one v-SNARE is called synaptobrevin. Botulinum B toxin, one of the most lethal toxins known and the most serious cause of food poisoning, contains a protease that binds synaptobrevin, thus inhibiting release of acetylcholine at the neuromuscular junction and often proving fatal.

Step 7: Fusion of the vesicle with the acceptor membrane occurs once the v- and t-SNARES are closely aligned. After vesicle fusion and release of contents, GTP is hydrolyzed to GDP, and the Rab·GDP molecules are released into the cytosol. When a SNARE on one membrane interacts with a SNARE on another membrane, linking the two, this is referred to as a trans-SNARE complex or a SNARE pin. Interactions of SNARES on the same membrane form a cis-SNARE complex. In order to dissociate the four-helix bundle between the v- and t-SNARES so that they can be reused, two additional proteins are required. These are an ATPase (NSF) and a-SNAP. NSF hydrolyzes ATP and the energy released dissociates the four helix bundle making the SNARE proteins available for another round of membrane fusion.

Step 8: Certain components, such as the Rab and SNARE proteins, are recycled for subsequent rounds of vesicle fusion.

During the above cycle, SNARES, tethering proteins, Rab, and other proteins all collaborate to deliver a vesicle and its contents to the appropriate site.

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