摘要：

In the early secretory pathway newly synthesized proteins are transported sequentially through the endoplasmic reticulum (ER), vesicular-tubular clusters (VTCs) and the Golgi complex. Traffic between these compartments is mediated by vesicular and/or tubular carriers that tether and fuse with their appropriate target membrane. Membrane fusion is promoted by SNARE complexes. When this work began, only the exocytic SNARE complexes were characterized in detail; the structural features shared by SNARE complexes from different fusion steps were not known. I contributed in characterizing the subunit structure, assembly and regulation of a quaternary SNARE complex that mediates early ER-to-Golgi transport. Recombinant syntaxin 5, membrin and rbet1, three Q-SNAREs, assemble cooperatively to create a high affinity binding site for sec22b, an R-SNARE. This organization is remarkably similar to the synaptic complex, suggesting a common pattern for all transport steps. The syntaxin 5 amino terminal domain potently inhibits SNARE complex assembly. Interestingly, although sec22b binds to the combination of syntaxin 5, membrin and rbet1, it can only bind if present while the other three assemble; sec22b cannot bind to a pre-assembled ternary complex, suggesting a potentially important regulatory step in the specific fusion event(s) this SNARE complex catalyzes. Finally, the quaternary complex is demonstrated to be a bona fide species in living cells. COPII-coated vesicles deliver secretory cargo from ER exit sites to VTCs in the initial steps of the secretory pathway. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. I documented in vitro direct tethering and SNARE-dependent fusion of ER-derived COPII transport vesicles to form larger cargo containers. The assembly did not require Golgi membranes, pre-existing VTCs, or COPI function. Thus, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles. My biochemical reconstitution provided direct evidence that the first membrane fusion event in the secretory pathway is the homotypic fusion of COPII vesicles and paves the way for future mechanistic studies of ER/Golgi SNAREs.

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