Genetic interactions with the yeast Q-SNARE VTI1 reveal novel functions for the R-SNARE YKT6

SNARE proteins are required for fusion of transport vesicles with target me mbranes. Previously, we found that the yeast Q-SNARE Vti1p is involved in t ransport to the cis-Golgi, to the prevacuole/late endosome, and to the vacu ole. Here we identified a previously uncharacterized gene, VTS1, and the R- SNARE YKT6 both as multicopy and as low copy suppressors of the growth and vacuolar transport defect in uti1-2 cells. Ykt6p was known to function in r etrograde traffic to the cis-Golgi and homotypic vacuolar fusion. We found that VTI1 and YKT6 also interacted in traffic to the prevacuole and vacuole , indicating that these SNARE complexes contain Ykt6p, Vti1p, plus Pep12p a nd Ykt6p, Vti1p, Vam3p, plus Vam7p, respectively. As Ykt6p was required for several transport steps, R-SNAREs cannot be the sole determinants of speci ficity. To study the role of the 0 layer in the SNARE motif, we introduced the mutations vti1-Q158R and ykt6-R165Q. SNARE complexes to which Ykt6p con tributed a fourth glutamine residue in the 0 layer were nonfunctional, sugg esting an essential function for arginine in the 0 layer of these complexes . vti1-Q158R cells had severe defects in several transport steps, indicatin g that the second arginine in the 0 layer interfered with function.