Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal H(abc) domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function.
Department of Biochemistry, University of Washington, Seattle, WA 98195-3750, USA.
Braden T
BT
Lobingier
Alexey J
AJ
Merz
D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D051938:Munc18 Proteins / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D008565:Membrane Proteins; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; C497931:SEC1 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; C107706:VAM3 protein, S cerevisiae; C494205:VAM7 protein, S cerevisiae; C065454:VPS33 protein, S cerevisiae; D033921:Vesicular Transport Proteins