Phosphorylation of the autoinhibitory domain of the Sso t-SNAREs promotes binding of the Vsm1 SNARE regulator in yeast.
We have shown that protein kinase A phosphorylation of t-SNAREs inhibits SNARE assembly and suppresses endo- and exocytosis in yeast. Herein, we show that protein kinase A phosphorylation of the Sso exocytic t-SNAREs promotes the binding of Vsm1, a potential SNARE regulator identified previously in our laboratory. Phosphorylation of Sso increases its affinity for Vsm1 by more than fivefold in vitro and both phosphorylated Sso1, as well as Sso1 bearing an aspartate substitution at position 79, interact tightly with Vsm1. Vsm1 binding is dependent upon the NH2-terminal autoinhibitory domain of Sso, and constitutively "open" forms of the t-SNARE show a reduction in Vsm1 binding in vivo. The substitution of serine-79 in Sso1 with an alanine residue or the treatment of yeast with C2-ceramide, which results in the dephosphorylation of serine-79, both inhibit Vsm1 binding in vivo. Importantly, Vsm1 binding to Sso seems to preclude Sso binding to its partner t-SNARE, Sec9, and vice versa. This is consistent with the idea that Vsm1 is an inhibitor of SNARE assembly in yeast. Thus, one way by which phosphorylation inhibits SNARE assembly could be by regulating the association of inhibitory factors that control the ability of t-SNAREs to form complexes in vivo.
Marash
Michael
M
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Gerst
Jeffrey E
JE
D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004705:Endocytosis; D005089:Exocytosis; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D033921:Vesicular Transport Proteins
C108125:DDI1 protein, S cerevisiae; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; C084006:SSO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D017868:Cyclic AMP-Dependent Protein Kinases