Membrane docking and fusion are the terminal events in vesicular transport. We have used the yeast vacuole as a model system to probe SNARE-mediated membrane fusion events in general, and fusion events in the endolysosomal system in particular.
During SNARE-mediated fusion, the two membranes are first brought into proximity. Membrane-anchored SNARE proteins then wind together in a concerted oligomerization and folding reaction — "zippering" — which leads to the formation of a trans-SNARE complex. Further zippering drives the membranes into contact, followed by merger of the two bilayers, fusion pore opening, and content mixing.
After fusion, the extremely stable cis-SNARE complex is disassembled through the action of an ATP-dependent chaperone system (Sec17 and Sec18), generating uncomplexed SNAREs that are re-energized for another round of fusion.
By preparing a series of truncations of a soluble SNARE, Vam7 (Qc), we have been able to slow and then stop an incipient fusion reaction, trapping the SNARE complex in its assembled and poised pre-fusion state. These powerful and extremely potent (apparent Km ~1 nM) reagents are being used to understand the composition and topology of the pre-fusion complex, and to deliver optical probes into the heart of the fusion machine.
Of course, these cartoons are radical over-simplifications of docking and fusion as they occur in living cells. For each fusion reaction there are an array of other proteins: Rab small G proteins, Rab-binding tethering factors, assembly co-chaperones of the SM (Sec1-Munc18) family, and other factors including lipid and protein kinases.
Much of the last several years' work in our lab (and many others) has been dedicated to understanding the functions of SNARE cofactors. We have focused most intently on the Vps-C tethering complexes, CORVET and HOPS. These complexes have overlapping subunit compositions, and operate at the late endosome and lysosome, respectively.
We have demonstrated that a pair of subunits unique to each complex serve as Rab-binding modules, while a common subunit, the SM protein Vps33, engages both individual SNARE domains and assembled (or partially assembled) SNARE complexes. In addition, there are accessory binding sites within the Vps-C complex which engage the N-terminal domains of at lest two of the SNAREs.
Going forward, we are addressing several questions:
- What is the mechanistic role of SM proteins in SNARE assembly and function?
- What is the relationship between SNARE zippering and fusion pore opening at sub-millisecond time scales?
- How are incipient fusion pores stabilized? What keeps them from collapsing?
- Does quality control occur in SNARE assembly? Are incorrect complexes inactivated, disassembled, or both? If so, what are the mechanisms through which this occurs?
- How is the process of vesicle formation coupled to vesicle docking and fusion at target membranes?