Functional analysis of conserved structural elements in yeast syntaxin Vam3p

Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is c omposed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR). Surprisingly, we find that the N-t erminal domain of Vam3p is not essential for fusion, although analogous dom ains in other syntaxins are indispensible for fusion and/or protein-protein interactions. In contrast to the N-terminal domain, mutations in the SNARE motif of Vam3p or replacement of the SNARE motif of Vam3p with the SNARE m otif from other syntaxins inhibited fusion. Furthermore, the precise distan ce between the SNARE motif and the TMR was critical for fusion. Insertion o f only three residues after the SNARE motif significantly impaired fusion a nd insertion of 12 residues abolished fusion. As judged by co-immunoprecipi tation experiments, the SNARE motif mutations and the insertions did not al ter the association of Vam3p with Vam7p, Vti1p, Nyv-1p, and Ykt6p, other va cuolar SNARE proteins implicated in fusion. In contrast, the SNARE motif su bstitutions interfered with the stable formation of Vam3p complexes with Ny v1p and Vti1p, although Vam3p complexes with Vam7p and Ykt6p were still pre sent. Our data suggest that in contrast to previously characterized syntaxi ns, Vam3p contains only two domains essential for fusion, the SNARE motif a nd the TMR, and these domains have to be closely coupled to function in fus ion.