As a prelude to fusion, the R-SNARE on one membrane zippers with Qa-, Qb-, and Qc-SNAREs from its apposed fusion partner, forming a four-helical bundle that draws the two membranes together. Because Qa- and Qb-SNAREs are anchored to the same membrane and are adjacent in the 4-SNARE bundle, their two anchors might be redundant. Using the recombinant pure protein catalysts of yeast vacuole fusion, we now report that the specific distribution of transmembrane (TM) anchors on the Q-SNAREs is critical for efficient fusion. A TM anchor on the Qa-SNARE supports rapid fusion even when the other two Q-SNAREs are unanchored, while a TM anchor on the Qb-SNARE is dispensable and is insufficient for rapid fusion as the sole Q-SNARE anchor. This does not depend on which specific TM domain is attached to the Qa-SNARE but rather is due to the Qa-SNARE being anchored per se. The need for Qa-SNARE anchoring is even seen when the homotypic fusion and vacuole protein sorting protein (HOPS), the physiological catalyst of tethering and SNARE assembly, is replaced by an artificial tether. The need for a Qa TM anchor is thus a fundamental property of vacuolar SNARE zippering-induced fusion and may reflect the need for the Qa juxtamembrane (JxQa) region to be anchored between its SNARE and TM domains. This requirement for Qa-SNARE anchoring and correct JxQa position is bypassed by Sec17/Sec18, exploiting a platform of partially zippered SNAREs. Because Qa is the only synaptic Q-SNARE with a TM anchor, the need for Qa-specific anchoring may reflect a general requirement for SNARE-mediated fusion.

Rab GTPases, their effectors, SNAREs of the R, Qa, Qb, and Qc families, and SM SNARE-binding proteins catalyze intracellular membrane fusion. At the vacuole/lysosome, they are integrated by the homotypic fusion and vacuole protein sorting (HOPS) complex. Two HOPS subunits bind vacuolar Rabs for tethering, another binds the Qc SNARE, and a fourth HOPS subunit, an SM protein, has conserved grooves that bind R- and Qa-SNARE domains. Spontaneous quaternary SNARE complex assembly is very slow. We report an assay of SNARE complex assembly that does not rely on fusion and for which tethering does not coenrich the four SNAREs. HOPS is required in this assay for rapid SNARE complex assembly. Optimal assembly needs HOPS, lipid membranes to which the R- or Qa-SNARE and Ypt7:GTP are integrally bound, and each of the other three SNAREs. Each SNARE assembles into this complex relying on the others, suggesting four-SNARE complex assembly rather than direct binding of each to HOPS. SNAREs can be disassociated by Sec 17/Sec 18/ATP, completing a catalyzed cycle of SNARE assembly and disassembly.