Screening for suppressors of temperature sensitivity in a yeast mutant defective in vacuolar protein degradation

Abstract

Vps33p is a member of the Sec1/munc18-like protein (SM protein) family involved in vesicular protein transport to the yeast vacuole. It is part of a high molecular weight complex which is required for homotypic vacuole fusion, and participates in Golgi-to-endosome and endosome-to-vacuole transport steps. Deletions in the vps33 gene result in severe vacuolar protein sorting and vacuolar morphology defects. We used a temperature sensitive (ts) vps33 deletion strain in a high copy plasmid suppressor approach to identify genes possibly acting along the same vesicular trafficking pathway(s) as Vps33p. While only the original VPS33 gene could restore the vacuolar enzyme sorting and vacuolar morphology defects, several suppressors of temperature sensitivity were found. Sequence analysis identified the ubiquitin-ligase Ufd4p as the only open reading frame (ORF) of two suppressor plasmids. Further suppressor candidates included the ubiquitin-processing protease Ubp10p and genes whose products are involved in more general stress responses. The result of this screening supports the emerging concept of a crosstalk between vesicular trafficking pathways and the ubiquitin/proteasome system of protein degradation.