N-terminal domain of vacuolar SNARE Vam7p promotes trans-SNARE complex assembly.
SNARE-dependent membrane fusion in eukaryotic cells requires that the heptad-repeat SNARE domains from R- and Q-SNAREs, anchored to apposed membranes, assemble into four-helix coiled-coil bundles. In addition to their SNARE and transmembrane domains, most SNAREs have N-terminal domains (N-domains), although their functions are unclear. The N-domain of the yeast vacuolar Qc-SNARE Vam7p is a binding partner for the homotypic fusion and vacuole protein sorting complex (a master regulator of vacuole fusion) and has Phox homology, providing a phosphatidylinositol 3-phosphate (PI3P)-specific membrane anchor. We now report that this Vam7p N-domain has yet another role, one that does not depend on its physical connection to the Vam7p SNARE domain. By attaching a transmembrane anchor to the C terminus of Vam7p to create Vam7tm, we bypass the requirement for the N-domain to anchor Vam7tm to reconstituted proteoliposomes. The N-domain of Vam7tm is indispensible for trans-SNARE complex assembly in SNARE-only reactions. Introducing Vam7(1-125)p as a separate recombinant protein suppresses the defect caused by N-domain deletion from Vam7tm, demonstrating that the function of this N-domain is not constrained to covalent attachment to Vam7p. The Vam7p N-domain catalyzes the docking of apposed membranes by promoting transinteractions between R- and Q-SNAREs. This function of the Vam7p N-domain depends on the presence of PI3P and its affinity for PI3P. Added N-domain can even promote SNARE complex assembly when Vam7 still bears its own N-domain.
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA. hao.xu@usm.edu
Hao
H
Xu
William T
WT
Wickner
D001483:Base Sequence; D017931:DNA Primers; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; C494205:VAM7 protein, S cerevisiae