Abstract
Membrane fusion requires tethers, SNAREs of R, Qa, Qb, and Qc families, and chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. SNAREs have N-domains, SNARE domains that zipper into 4-helical RQaQbQc coiled coils, a short juxtamembrane (Jx) domain, and (often) a C-terminal transmembrane anchor. We reconstitute fusion with purified components from yeast vacuoles, where the HOPS protein combines tethering and SM functions. The vacuolar Rab, lipids, and R-SNARE activate HOPS to bind Q-SNAREs and catalyze trans-SNARE associations. With SNAREs initially disassembled, as they are on the organelle, we now report that R- and Qa-SNAREs require their physiological juxtamembrane (Jx) regions for fusion. Swap of the Jx domain between the R- and Qa-SNAREs blocks fusion after SNARE association in trans. This block is bypassed by either Sec17, which drives fusion without requiring complete SNARE zippering, or transmembrane-anchored Qb-SNARE in complex with Qa. The abundance of the trans-SNARE complex is not the sole fusion determinant, as it is unaltered by Sec17, Jx swap, or the Qb-transmembrane anchor. The sensitivity of fusion to Jx swap in the absence of a Qb transmembrane anchor is inherent to the SNAREs, because it remains when a synthetic tether replaces HOPS.