The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface.

The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Delta mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Delta mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Delta mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Delta mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.

Mapping of sporulation-specific functions in the yeast syntaxin gene SSO1.

The yeast Saccharomyces cerevisiae has two closely related plasma membrane syntaxins, Sso1p and Sso2p, which together provide an essential function in vegetative cells. However, Sso1p is also specifically needed during sporulation; and this function cannot be provided by Sso2p. We used fusions between SSO1 and SSO2 to map the sporulation-specific function of SSO1. We found that the two N-terminal alpha-helices Ha and Hb of Sso1p are important for sporulation, since it is reduced 8-fold for fusions where Ha and Hb are derived from Sso2p. In contrast, the C-terminal half of Sso1p does not seem to be specifically required for sporulation. Surprisingly, we further found that the 3' untranslated region (3'UTR) of SSO1 is essential for sporulation. Western blots failed to reveal a preferential expression of Sso1p in sporulating cells, indicating that effects on gene expression are unlikely to explain why the SSO1 3'UTR is needed for sporulation.

Characterization of temperature-sensitive mutations in the yeast syntaxin 1 homologues Sso1p and Sso2p, and evidence of a distinct function for Sso1p in sporulation.

The duplicated genes SSO1 and SSO2 encode yeast homologues of syntaxin 1 and perform an essential function during fusion of secretory vesicles at the plasma membrane. We have used in vitro mutagenesis to obtain a temperature-sensitive SSO2 allele, sso2-1, in which a conserved arginine has been changed to a lysine. A yeast strain that lacks SSO1 and carries the sso2-1 allele ceases growth and accumulates secretory vesicles at the restrictive temperature. Interestingly, the strain also has a pronounced phenotype at the permissive temperature, causing a defect in bud neck closure that prevents separation of mother and daughter cells. The same mutation was introduced into SSO1, producing the sso1-1 allele, which also has a temperature-sensitive phenotype, although less pronounced than sso2-1. A screen for high copy number suppressors of sso2-1 yielded three genes that are involved in the terminal step of secretion: SNC1, SNC2 and SEC9. The sso1-1 mutation interacts synthetically with a disruption of the MSO1 gene, which encodes a Sec1p interacting protein. Interestingly, we further found that both MSO1 and SSO1, but not SSO2, are required for sporulation. This difference is not due to differential expression, since SSO2 expressed from the SSO1 promoter failed to restore sporulation. We conclude that a functional difference exists between the Sso1 and Sso2 proteins, with the former being specifically required during sporulation.