Abstract
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.
Highlights
Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qcfamily SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex
Intracellular membrane fusion is catalyzed by families of fusion proteins which are conserved from yeast to humans and among the organelles (Wickner and Rizo, 2017)
These include Rab-family GTPases, large tethering complexes which bind to Rabs (Baker and Hughson, 2016), membrane-anchored SNARE proteins (Jahn and Scheller, 2006), and SNARE chaperones of the SM, Sec17/αSNAP and
Summary
With either the R- or the 3 Q-SNAREs. The Qc SNARE was either wildtype with its full-length SNARE domain or Qc3 which lacks the C-terminal 4 heptads of its SNARE domain. When the 3Q complex includes wild-type full-length Qc, HOPS-mediated fusion (Figure 1B , black curve) shows only minor effects from adding either Sec (red), Sec with a nonhydrolyzable ATP analog (blue), or Sec, Sec, and ATP S (green). The dimeric tether GST-PX (Song and Wickner, 2019) supports fusion with pre-assembled wild-type Q-SNAREs (Figure 1D , black curve), but this fusion is blocked by Sec (Figure 1D , red curve) without rescue by Sec (green curve). Full-length SNAREs support zippering and fusion with either the HOPS or GST-PX tether, a kinetic intermediate accumulates which gives some additional fusion upon addition of Sec17/Sec18/ATP S (Figure 1, B and D , black curves). Fusion intermediate accumulated, since there is rapid fusion upon their addition
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