Direct Involvement of Phosphatidylinositol 4-Phosphate in Secretion in the Yeast Saccharomyces cerevisiae*
- From the ‡Department of Biology, Utah State University, Logan, Utah 84322-5305 and the §Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202
Abstract
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 °C and that sec14-3 cells could grow at an otherwise nonpermissive temperature (34 °C) when carrying a plasmid overexpressingPIK1, 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 differentpik1 ts mutants were examined. Upon shift to restrictive temperature (37 °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.
Footnotes
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↵* This work was supported in part by Grant MCB 9630108 from the National Science Foundation (to D. B. D.) and by funds from the Utah Agricultural Experiment Station (to D. B. D. and J. Y. T.), Eli Lilly and Co. (to J. Y. T.), in part by United States Public Health Service Predoctoral Traineeship GM07232 (to E. A. S.), and by National Institutes of Health Research Grant GM21841 from the National Institute for General Medical Sciences (to J. T.). This is Utah Agricultural Experiment Station paper number 7195.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Dept. of Biology, Utah State University, Logan, UT 84322-5305. Tel.: 435-797-3711; Fax: 435-797-1575; E-mail: dewald@biology.usu.edu.
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↵2 In many publications, including our own (19), the genotype of CTY1-1A has been designated as sec14-1. However, the sec14 allele in CTY1-1A was derived from strain SF292-2A and was originally designated sec14-3 (11). Hence, the latter designation (sec14-3) has been adopted here.
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↵3 E. Harsay and R. W. Schekman, personal communication.
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↵4 H. Hama and D. B. DeWald, unpublished results.
- Abbreviations:
- ER
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endoplasmic reticulum
- DAG
-
diacylglycerol
- HPLC
-
high performance liquid chromatography
- M(IP)2C
-
mannosyldiinositolphosphorylceramide
- PITP
-
phosphatidylinositol transfer protein
- PtdCho
-
phosphatidylcholine
- PtdIns
-
phosphatidylinositol
- PtdIns(3)P
-
phosphatidylinositol 3-phosphate
- PtdIns(4)P
-
phosphatidylinositol 4-phosphate
- PtdIns(4
-
5)P2, phosphatidylinositol 4,5-bisphosphate
- PtdIns(3
-
5)P2, phosphatidylinositol 3,5- bisphosphate
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- Received July 19, 1999.
- Revision received August 14, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
