Direct involvement of phosphatidylinositol 4-phosphate in secretion in the yeast Saccharomyces cerevisiae.
The SEC14 gene encodes an essential phosphatidylinositol (PtdIns) transfer protein required for formation of Golgi-derived secretory vesicles in yeast. Suppressor mutations that rescue temperature-sensitive sec14 mutants provide an approach for determining the role of Sec14p in secretion. One suppressor, sac1-22, causes accumulation of PtdIns(4)P. SAC1 encodes a phosphatase that can hydrolyze PtdIns(4)P and certain other phosphoinositides. These findings suggest that PtdIns(4)P is limiting in sec14 cells and that elevation of PtdIns(4)P production can suppress the secretory defect. Correspondingly, we found that PtdIns(4)P levels were decreased significantly in sec14-3 mutants shifted to 37 degrees C and that sec14-3 cells could grow at an otherwise nonpermissive temperature (34 degrees C) when carrying a plasmid overexpressing PIK1, encoding one of two essential PtdIns 4-kinases. This effect is specific because overexpression of the other PtdIns 4-kinase gene (STT4) or a PtdIns 3-kinase gene (VPS34) did not rescue sec14-3 cells. To further address Pik1p function in secretion, two different pik1(ts) mutants were examined. Upon shift to restrictive temperature (37 degrees C), the PtdIns(4)P levels dropped by about 60% in both pik1(ts) strains within 1 h. During the same period, cells displayed a reduction (40-50%) in release of a secreted enzyme (invertase). However, similar treatment did not effect maturation of a vacuolar enzyme (carboxypeptidase Y). These findings indicate that, first, PtdIns(4)P limitation is a major contributing factor to the secretory defect in sec14 cells; second, Sec14p function is coupled to the action of Pik1p, and; third, PtdIns(4)P has an important role in the Golgi-to-plasma membrane stage of secretion.
Hama
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Department of Biology, Utah State University, Logan, Utah 84322-5305, USA.
Schnieders
E A
EA
Thorner
J
J
Takemoto
J Y
JY
DeWald
D B
DB
D019870:1-Phosphatidylinositol 4-Kinase / Q000235:genetics / Q000502:physiology; D001692:Biological Transport; D002268:Carboxypeptidases / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D043402:Cathepsin A; D015966:Gene Expression Regulation, Fungal; D006026:Glycoside Hydrolases / Q000378:metabolism; D008565:Membrane Proteins; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014617:Vacuoles / Q000378:metabolism; D043324:beta-Fructofuranosidase
D002352:Carrier Proteins; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D049294:Phospholipid Transfer Proteins; C483438:SEC14 protein, S cerevisiae; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C037178:phosphatidylinositol 4-phosphate; D019870:1-Phosphatidylinositol 4-Kinase; C441760:PIK1 protein, S cerevisiae; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D002268:Carboxypeptidases; D043402:Cathepsin A