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首页> 外文期刊>Molecular and Cellular Biology >Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.
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Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.

机译：酿酒酵母中磷脂酰肌醇特异性磷脂酶C的遗传和生化特征。

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Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses.
机译：磷脂酰肌醇特异性磷脂酶C（PI-PLC）水解磷脂酰肌醇4,5-双磷酸酯（PIP2）产生两个第二信使，肌醇1,4,5-三磷酸酯和1,2-二酰基甘油。聚合酶链反应用于分离酿酒酵母基因（PLC1），该基因编码869个氨基酸的蛋白质（命名为Plc1p），就整体序列相似性和结构域排列而言，它与哺乳动物PI-PLC的δ亚型最相似。 Plc1p包含在所有高等真核PI-PLC中发现的保守X和Y结构域（分别与大鼠delta 1 PI-PLC的相应结构域具有51％和29％的同一性），并且还包含一个推测的Ca（2+）结合位点（EF手形图案）。 Plc1p，通过在其氨基末端附近的His6区域和c-myc表位的框内插入修饰，从GAL1启动子中过表达，通过镍螯合亲和色谱法部分纯化，显示其是一种具有特性的体外活性PLC酶类似于哺乳动物。 Plc1p活性严格依赖于Ca2 +：在高Ca2 +浓度（0.1 mM）下，该酶以比磷脂酰肌醇更快的速率水解PIP2，而在低Ca2 +浓度（0.5 microM）下仅水解PIP2。携带两种不同的缺失插入突变（plc1 delta 1 :: HIS3和plc1 delta 2 :: LEU2）的细胞是可行的，但显示出几种独特的表型，包括温度敏感型生长（在35摄氏度以上可行），渗透敏感性和在允许温度（23至30摄氏度）下利用半乳糖，棉子糖和甘油存在缺陷。此处报道的发现表明，酿酒酵母中PIP2的水解是许多营养和应激相关反应所必需的。

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