t-SNARE dephosphorylation promotes SNARE assembly and exocytosis in yeast.
The role of protein phosphorylation in secretion is not well understood. Here we show that yeast lacking the Snc1,2 v-SNAREs, or bearing a temperature-sensitive mutation in the Sso2 t-SNARE, are rescued at restrictive conditions by the addition of ceramide precursors and analogs to the growth medium. Rescue results from dephosphorylation of the Sso t-SNAREs by a ceramide-activated type 2A protein phosphatase (Sit4) involved in cell cycle control. Sso t-SNARE dephosphorylation correlated with its assembly into complexes with the Sec9 t-SNARE, both in vitro and in vivo, and with an increase in protein trafficking and secretion in cells. SNARE complexes isolated under these conditions contained only Sso and Sec9, suggesting that a t-t-SNARE fusion complex is sufficient to confer exocytosis. Mutation of a single PKA site (Ser79 to Ala79) in Sso1 resulted in a decrease in phosphorylation and was sufficient to confer growth to snc cells at restrictive conditions. Thus, modulation of t-SNARE phosphorylation regulates SNARE complex assembly and membrane fusion in vivo.
Marash
M
M
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Gerst
J E
JE
D005089:Exocytosis; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D008565:Membrane Proteins; C064769:N-acetylsphingosine; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; C084007:SSO2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D010749:Phosphoprotein Phosphatases; D013110:Sphingosine