Genetic Interactions with the Yeast Q-SNARE VTI1Reveal Novel Functions for the R-SNARE YKT6

Meik Dilcher,Beate Köhler,Gabriele Fischer Von Mollard

doi:10.1074/jbc.m101551200

Abstract

SNARE proteins are required for fusion of transport vesicles with target membranes. Previously, we found that the yeast Q-SNARE Vti1p is involved in transport to the cis-Golgi, to the prevacuole/late endosome, and to the vacuole. 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 vti1-2 cells. Ykt6p was known to function in retrograde 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 and Ykt6p, Vti1p, Vam3p, plus Vam7p, respectively. As Ykt6p was required for several transport steps, R-SNAREs cannot be the sole determinants of specificity. 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 contributed a fourth glutamine residue in the 0 layer were nonfunctional, suggesting an essential function for arginine in the 0 layer of these complexes. vti1-Q158R cells had severe defects in several transport steps, indicating that the second arginine in the 0 layer interfered with function.

Highlights

Transport between different organelles is mediated by transport vesicles, which bud from the donor compartment [1]
These transport steps are blocked in vti1–2 cells at the non-permissive temperature, whereas transport to the Golgi is not affected [9, 20]. vti1–2 cells display a growth defect at 37 °C, which we utilized for a multicopy (2 ␮m) suppressor screen
The R-SNARE Ykt6p is encoded by this DNA fragment

Summary

Introduction

Transport between different organelles is mediated by transport vesicles, which bud from the donor compartment [1]. Vti1p and the syntaxin-related endosomal Q-SNARE Pep12p are the only SNAREs identified so far in transport from the Golgi to the prevacuolar/late endosomal compartment [9, 13]. This transport pathway is used by many vacuolar proteins, for example by carboxypeptidase Y (CPY). We investigated the role of the amino acid residues in the 0 layer of Ykt6p and Vti1p These SNARE complexes are nonfunctional with four glutamine residues and defective with two arginine and two glutamine residues in the 0 layer

Objectives

Results

Conclusion