Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering

Abstract

Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/aSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 will drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE which face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

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

“…A "trans-20s" (Song et al, 2021;Rizo et al, 2021) may generally drive fusion, since its elements are common to all exocytic and endocytic trafficking while the tethering complexes of other organelles are varied and often do not have the organelle's SM protein as a tightly-bound subunit like Vps33 is in HOPS (Baker and Hughson, 2016). These findings support the model (Song et al, 2021) that HOPS only acts for tethering and to catalyze the initial phase of zippering in a Sec17-resistant manner. HOPS is specific for fusion at the vacuole/lysosome, but Sec17/αSNAP, Sec18/NSF, and SNAREs are general elements of exocytic and endocytic vesicular trafficking.…”

“…Once SNAREs are partially zippered in trans, Sec17 and Sec18 do not need HOPS to support the completion of fusion. The completion of SNARE zippering is promoted by Sec17 (Song et al, 2021), and Sec17 displaces HOPS from SNARE complexes (Collins et al, 2005;Schwartz et al, 2017). The interactions among the SNAREs, Sec17/αSNAP and Sec18/NSF are seen at a molecular level in the 20s complex, consisting of a 4-SNARE coiled coil anchored to membranes at their C-termini, surrounded by up to 4 Sec17/αSNAP molecules , and all capped at the membrane-distal end by Sec18/NSF (Zhao, 2015).…”

“…Reagents were purchased, and proteins purified, as described in Song et al, 2021. GST-PX constructions.…”

“…For GST-PX F AGGGCGCCATGGATCCGGCAGCTAATTCTGTAGGGAA R AGTTGAGCTCGTCGACTATGGCTTTGACAACTGCAGGA GST-PX was prepared as described (Fratti and Wickner, 2007). Proteoliposome preparation and fusion assays were as described in Song et al (2021). In brief, proteoliposomes were separately preincubated for 10 min at 27 o C with EDTA and GTP, followed by addition of MgCl 2 , to load the Ypt7 with GTP.…”

“…The ubiquitous chaperones Sec17 and Sec18 will bind to partially zippered SNARE complexes and use the N-terminal apolar loop on Sec17 to trigger fusion without needing complete zippering (Schwartz and Merz, 2009;Zick et al, 2015;Schwartz et al, 2017;Song et al, 2021). Sec17 has direct affinity for HOPS (Song et al, 2021); it has been unclear whether HOPS is needed for Sec17 and Sec18 to engage partially zippered SNAREs and mediate zippering-bypass fusion. We now exploit a synthetic tether to show that HOPS is not required for Sec17 and Sec18 to drive zippering-bypass fusion.…”