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 membrane to initiate budding (Figure 13–14). Other GEFs and coat-recruitment GTPases operate in a similar way on other membranes.
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 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.
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
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.