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. 2015 Sep 4;349(6252):1111-4.
doi: 10.1126/science.aac7906.

A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly

Richard W Baker  1 , Philip D Jeffrey  1 , Michael Zick  2 , Ben P Phillips  1 , William T Wickner  2 , Frederick M Hughson  3
Affiliations

A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly

Richard W Baker et al. Science. .

Abstract

Fusion of intracellular transport vesicles requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18-family (SM) proteins. Membrane-bridging SNARE complexes are critical for fusion, but their spontaneous assembly is inefficient and may require SM proteins in vivo. We report x-ray structures of Vps33, the SM subunit of the yeast homotypic fusion and vacuole protein-sorting (HOPS) complex, bound to two individual SNAREs. The two SNAREs, one from each membrane, are held in the correct orientation and register for subsequent complex assembly. Vps33 and potentially other SM proteins could thus act as templates for generating partially zipped SNARE assembly intermediates. HOPS was essential to mediate SNARE complex assembly at physiological SNARE concentrations. Thus, Vps33 appears to catalyze SNARE complex assembly through specific SNARE motif recognition.

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Figures

Fig. 1
Fig. 1. Structure of the Qa-SNARE Vam3 bound to the SM protein Vps33
(A) Shown are the C. thermophilum SNAREs whose SNARE motifs were co-crystallized with Vps33–Vps16. Cross-hatching indicates regions that were disordered in the resulting crystal structures (see also Fig. 2). TMD, transmembrane domain. (B) Vps33–Vps16 crystal structure, highlighting domains 1 (red), 2 (green), and 3 (blue). (C) Complex containing the Vam3 SNARE motif (see fig. S2). The zero-layer Q is highlighted. Vps16 is present in this structure but is omitted for clarity. (D and E) Comparison between Vps33–Vam3 and Munc18a–syntaxin1a (PDB code 3C98) complexes (see fig. S4).
Fig. 2
Fig. 2. Binding to a conserved surface groove on Vps33 orients and aligns the R-SNARE Nyv1 for assembly
(A) Vps33–Nyv1 crystal structure (Vps16 omitted for clarity; see fig. S3). Red indicates mutations employed in Fig. 3. (B) The Nyv1 binding site is conserved (as calculated by ConSurf ()) among Vps33 homologs. The same site, indicated with cyan spheres, is conserved among Munc18 homologs (shown is 3PUJ). (C) Vps33, Nyv1, and Vam3 form a ternary complex as judged by size exclusion chromatography. Nyv1 and Vam3 SNARE motifs contain an N-terminal maltose binding protein tag. (D) Model showing both SNARE domains binding simultaneously to Vps33.
Fig. 3
Fig. 3. Functional consequences of disrupting R-SNARE binding
(A) Yeast vacuolar morphology as visualized by FM4-64 staining (see fig. S8). Cells containing 1-3 vacuoles were classified as having wild-type morphology. All error bars in this figure represent mean ± SD (N = 3). (B-L) In vitro fusion assay using purified HOPS containing wild-type or mutant Vps33. Proteoliposomes show an increase in FRET signal upon fusion and mixing of their luminal contents. Control experiments employ PEG as an artificial tethering factor. (B-D) Vps33(G338E) HOPS is fusion deficient at low SNARE concentrations. (E and F) Wild-type and mutant HOPS support membrane tethering. (G-L) Vps33(G338E) HOPS is compromised in recruiting (G-I) full-length Vam7 and (J-L) Vam7ΔPX into fusogenic trans-SNARE complexes. (M) Model showing the Vps33-templated initiation of trans-SNARE complex assembly. Numbers refer to SNARE complex core layers (see fig. S1). The remaining subunits of the HOPS complex, as well as Ypt7, are omitted for clarity. Further experiments are needed to clarify whether the fully zippered SNAREs remain associated with HOPS.
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