Biology subjects

Wickner, W. T.

Publications and source records attributed to Wickner, W. T..

3 recordsLinked to original sources

bioRxiv2021

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/SNAP, 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.

biochemistry↗

bioRxiv2021

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

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/SNAP 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. Though Sec17 aids zippering, 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 to fusion, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

biochemistry↗

bioRxiv2019

HOPS recognizes each SNARE, assembling ternary trans-complexes for sudden fusion upon engagement with the 4th SNARE

Vacuole fusion requires SNAREs, Sec17/18, a Rab, and HOPS. We find that co-incubation of HOPS, proteoliposomes bearing the Rab and R-SNARE, and proteoliposomes with the Rab and any two Q-SNAREs yields a trans complex which includes these 3 SNAREs. The missing Q-SNARE then triggers a burst of fusion, indicating that each HOPS, R-, and QxQy-SNARE trans-complex is an activated intermediate for functional Qz-SNARE incorporation. HOPS can assemble activated fusion intermediates because it recognizes each of the four SNAREs, binding them independently. HOPS-dependent fusion is saturable for each Q-SNARE, indicating saturable functional sites on HOPS. Though a nonspecific tether allows fusion with pre-assembled Q-SNAREs, only HOPS catalyzes fusion when the Q-SNAREs are not pre-assembled by ushering each Q-SNARE into a functional complex. In contrast, there is little spontaneous functional assembly of the 3 Q-SNAREs. HOPS thus recognizes each of the 4 SNAREs to assemble a versatile set of activated fusion intermediates.

biochemistry↗