| Abstract: |
Transport between Golgi cisternae in a cell-free system is blocked by either nonhydrolyzable analogue of GTP (GTPγS) or by the sulfhydryl reagent N-ethylmaleimide (NEM). With GTPγS, vesicles accumulate encased in a coat which is frozen in place because GTP cannot be hydrolyzed. With NEM, uncoated vesicles accumulate docked to their target cisterna, but fail to fuse; NEM inactivates a protein required for fusion, termed NSF, for NEM Sensitive Fusion protein. The coat is composed of many copies of a protein termed coatomer (short for coat protomer), an assembly of seven distinct subunits, termed COP proteins. The coated vesicles also contain many copies of a GTPase, ARF. Cytosolic ARF binds GTP, binds to the Golgi, and then recruits cytosolic coatomer to join the assembling coat. Later, after budding, ARF hydrolyzes its bound GTP, releasing coatomer; this explains why non-hydrolyzable GTP blocks transport and accumulates coated transport vesicles. Binding of the fusion protein NSF to Golgi membranes requires Soluble NSF Attachment Proteins such as α-SNAP, or the neuron-specific homologue β-SNAP. NSF and SNAP proteins assemble on Golgi and other membranes by binding a SNAp REceptor, or SNARE complex, composed of one SNARE emanating from the transport vesicle (the v-SNARE) and another SNARE emanating from the target membrane (t-SNARE). Each type of transport vesicle appears to have its own particular v-SNARE, which appears to target it by binding the particular t-SNARE that marks its target membrane. NSF initiates the fusion process when it hydrolyzes ATP and disrupts the SNARE complex. The biophysical mechanisms involved are still unclear. The same general fusion machinery used for protein traffic in all cells also underlies the regulated exocytosis responsible for the controlled release of neurotransmitters and hormones. Synaptic vesicles (storing transmitter at nerve endings) and the plasma membrane of nerves each contain v- and t-SNAREs, respectively, that assemble with SNAPs and NSF; all of these components are now known to be required for synaptic transmission in vivo. The likely calcium sensor, the synaptic vesicle membrane protein synaptotagmin, is a v-SNARE specialized for binding the neuron-specific SNAP isoform β-SNAP (but not the general isoform α-SNAP) and the signal-regulated phosphoinositide PI-3,4,5-P3 in a calcium-dependent process. |
