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

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

Heterologous expression of syntaxin 6 in Saccharomyces cerevisiae

The molecular mechanisms of vesicular protein transport in eukaryotic cells are highly conserved. Members of the syntaxin family play a pivotal role in the membrane fusion process. We have expressed rat syntaxin 6 and its cytoplasmic domain in wild-type and pep12 mutant strains of Saccharomyces cerevisiae to elucidate the role of the syntaxin 6-dependent vesicular trafficking step in yeast. Immunofluorescence microscopy revealed a punctate, Golgi-like staining pattern for syntaxin 6, which only partially overlapped with Pep12p in wild-type yeast cells. In contrast to Pep12p, syntaxin 6 was not mislocalized to the vacuole upon expression from 2 micron vectors, which might be attributed to conserved sorting and retention signals. Syntaxin 6 was not capable of complementing the sorting and maturation defects of the vacuolar hydrolase CPY in pep12 null mutants. No dominant negative effects of either syntaxin 6 or syntaxin 6DC overexpression on CPY sorting and maturation were observed in wild-type yeast cells. We conclude that syntaxin 6 and Pep12p do not act at the same vesicular trafficking step(s) in yeast and higher eukaryotes