Hama H, Schnieders E A, Thorner J, Takemoto J Y, DeWald D B
Department of Biology, Utah State University, Logan, Utah 84322-5305, USA.
J Biol Chem. 1999 Nov 26;274(48):34294-300. doi: 10.1074/jbc.274.48.34294.
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.
SEC14基因编码一种必需的磷脂酰肌醇（PtdIns）转移蛋白，该蛋白是酵母中高尔基体衍生分泌囊泡形成所必需的。拯救温度敏感型sec14突变体的抑制突变提供了一种确定Sec14p在分泌中作用的方法。一种抑制因子sac1-22会导致PtdIns(4)P的积累。SAC1编码一种磷酸酶，可水解PtdIns(4)P和某些其他磷酸肌醇。这些发现表明PtdIns(4)P在sec14细胞中是有限的，并且PtdIns(4)P产量的升高可以抑制分泌缺陷。相应地，我们发现转移到37摄氏度的sec14-3突变体中PtdIns(4)P水平显著降低，并且当携带过表达PIK1的质粒时，sec14-3细胞可以在原本不允许生长的温度（34摄氏度）下生长，PIK1编码两种必需的磷脂酰肌醇4-激酶之一。这种效应是特异性的，因为另一种磷脂酰肌醇4-激酶基因（STT4）或磷脂酰肌醇3-激酶基因（VPS34）的过表达并不能拯救sec14-3细胞。为了进一步研究Pik1p在分泌中的功能，我们检测了两种不同的pik(ts)突变体。转移到限制温度（37摄氏度）后，两种pik(ts)菌株中的PtdIns(4)P水平在1小时内下降了约60%。在同一时期，细胞分泌的一种酶（转化酶）的释放减少了（40-50%）。然而，类似的处理对液泡酶（羧肽酶Y）的成熟没有影响。这些发现表明，第一，PtdIns(4)P的限制是sec14细胞分泌缺陷的一个主要促成因素；第二，Sec14p的功能与Pik1p的作用相关联；第三，PtdIns(4)P在从高尔基体到质膜的分泌阶段具有重要作用。
