t-SNARE phosphorylation regulates endocytosis in yeast.
Earlier we demonstrated that activation of a ceramide-activated protein phosphatase (CAPP) conferred normal growth and secretion to yeast lacking their complement of exocytic v-SNAREs (Snc1,2) or bearing a temperature-sensitive mutation in an exocytic t-SNARE (Sso2). CAPP activation led to Sso dephosphorylation and enhanced the assembly of t-SNAREs into functional complexes. Thus, exocytosis in yeast is modulated by t-SNARE phosphorylation. Here, we show that endocytic defects in cells lacking the v- and t-SNAREs involved in endocytosis are also rescued by CAPP activation. Yeast lacking the Tlg1 or Tlg2 t-SNAREs, the Snc v-SNAREs, or both, undergo endocytosis after phosphatase activation. CAPP activation correlated with restored uptake of FM4-64 to the vacuole, the uptake and degradation of the Ste2 receptor after mating factor treatment, and the dephosphorylation and assembly of Tlg1,2 into SNARE complexes. Activation of the phosphatase by treatment with C(2)-ceramide, VBM/ELO gene inactivation, or by the overexpression of SIT4 was sufficient to confer rescue. Finally, we found that mutation of single PKA sites in Tlg1 (Ser31 to Ala31) or Tlg2 (Ser90 to Ala90) was sufficient to restore endocytosis, but not exocytosis, to snc cells. These results suggest that endocytosis is also modulated by t-SNARE phosphorylation in vivo.
Gurunathan
Sangiliyandi
S
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Marash
Michael
M
Weinberger
Adina
A
Gerst
Jeffrey E
JE
D004705:Endocytosis / Q000502:physiology*; D005656:Fungal Proteins / Q000378:metabolism*; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases; D011726:Pyridinium Compounds / Q000378:metabolism; D050765:Qa-SNARE Proteins; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D050683:R-SNARE Proteins; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014157:Transcription Factors; D022161:Transport Vesicles / Q000378:metabolism; D015003:Yeasts
C092350:FM 4-64; D005656:Fungal Proteins; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C064769:N-acetylsphingosine; D011726:Pyridinium Compounds; D050765:Qa-SNARE Proteins; D000644:Quaternary Ammonium Compounds; D050683:R-SNARE Proteins; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; C083241:SNC1 protein, S cerevisiae; C083242:SNC2 protein, S cerevisiae; C084007:SSO2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C112252:TLG2 protein, S cerevisiae; D014157:Transcription Factors; D010770:Phosphotransferases; D010749:Phosphoprotein Phosphatases; D013110:Sphingosine