Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS

Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that fusion intermediates with arrested SNARE zippering, formed with a synthetic tether but without HOPS, support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.

Membrane Fusion can be Driven by Sec18/NSF, Sec17/αSNAP, and trans-SNARE complex without HOPS

Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that SNARE zippering-arrested fusion intermediates that are formed without HOPS support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.

The HOPS/class C Vps complex tethers membranes by binding to one Rab GTPase in each apposed membrane

Many Rab GTPase effectors are membrane-tethering factors, that is, they physically link two apposed membranes before intracellular membrane fusion. In this study, we investigate the distinct binding factors needed on apposed membranes for Rab effector–dependent tethering. We show that the homotypic fusion and protein-sorting/class C vacuole protein-sorting (HOPS/class C Vps) complex can tether low-curvature membranes, that is, liposomes with a diameter of ∼100 nm, only when the yeast vacuolar Rab GTPase Ypt7p is present in both tethered membranes. When HOPS is phosphorylated by the vacuolar casein kinase I, Yck3p, tethering only takes place when GTP-bound Ypt7p is present in both tethered membranes. When HOPS is not phosphorylated, however, its tethering activity shows little specificity for the nucleotide-binding state of Ypt7p. These results suggest a model for HOPS-mediated tethering in which HOPS tethers membranes by binding to Ypt7p in each of the two tethered membranes. Moreover, because vacuole-associated HOPS is presumably phosphorylated by Yck3p, our results suggest that nucleotide exchange of Ypt7p on multivesicular bodies (MVBs)/late endosomes must take place before HOPS can mediate tethering at vacuoles.