Asymmetric Rab activation of vacuolar HOPS to catalyze SNARE complex assembly

Intracellular membrane fusion requires Rab-family GTPases, their effector tethers, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, and SNARE chaperones of the Sec1/Munc18 (SM), Sec17/α-SNAP, and Sec18/NSF families. We have developed an assay using fluorescence resonance energy transfer to measure SNARE complex formation in real time. We now show that yeast vacuolar SNAREs assemble spontaneously into RQaQbQc complexes when the R- and Qa-SNAREs are concentrated in the same micelles or in cis on the same membrane. When SNAREs are free in solution or are tethered to distinct membranes, assembly requires catalysis by HOPS, the vacuolar SM and tethering complex. The Rab Ypt7 and vacuole lipids together allosterically activate the bound HOPS for catalyzing SNARE assembly, even if none of the SNAREs are membrane bound. HOPS-dependent fusion between proteoliposomes bearing R- or Qa-SNAREs shows a strict requirement for Ypt7 on the R-SNARE proteoliposomes but not on the Qa-SNARE proteoliposomes. This asymmetry is reflected in the strikingly different capacity of Ypt7 in cis to either the R- or Qa-SNARE to stimulate SNARE complex assembly. Membrane-bound Ypt7 activates HOPS to catalyze 4-SNARE complex assembly when it is on the same membrane as the R-SNARE but not the Qa-SNARE, thus explaining the asymmetric need for Ypt7 for fusion.