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 .