Phospholipid Metabolism and Membrane Function

酵母酿酒酵母对多种应激条件（例如，锌耗竭）， 调节几种酶活性的表达，包括参与磷脂合成的那些。 锌是藻生长和代谢所必需的营养元素。酿酒酵母和高等真核生物 细胞本申请中提出的工作将解决密钥的表达所通过的机制。 锌缺乏时，磷脂生物合成酶的活性受到调节。我们会研究 锌缺乏诱导胞浆Mg 2+依赖性和NEM敏感性磷脂酸的假说 磷酸酶，其降低ER中的磷脂酸浓度。这反过来又触发了Opilp介导的 抑制CHO 1（编码磷脂酰丝氨酸合酶）和其他UAS|不含基因。这 一个问题将解决使用磷酸酶突变体，将通过反向遗传方法分离 需要纯化胞质Mg 2+依赖性磷脂酸磷酸酶。的 将检测锌耗竭对该磷酸酶基因表达的调节，沿着 纯化的酶的生物化学性质。我们将检验E/C/7编码的假设， 乙醇胺激酶和CK/t编码的胆碱激酶响应于锌消耗而被诱导。 将研究锌介导的EKI 1和CKI 1基因调节的机制， 将使用选择性启动子突变检测每个基因调节的生物学相关性， 影响他们的表达。Opilp介导的CHO 1和Zaplp-1抑制的生物学相关性 PIS 1（编码磷脂酰肌醇合酶）的介导诱导将用以下突变体进行探索： 不能下调磷脂酰丝氨酸合酶或上调磷脂酰肌醇合酶活性， 分别响应于锌耗尽。我们将检验高- 亲合性质膜锌转运蛋白Zrt 1 p受磷脂组成变化的调节 由锌缺乏引起的磷脂合成调节引起。锌转运功能将 用磷脂合成缺陷的突变体和用重建成单层的Zrtlp来解决， 具有不同磷脂成分的囊泡。

The yeast Saccharomyces cerevisiae responds to a variety of stress conditions (e.g., zinc depletion) by regulating the expression of several enzyme activities including those involved in phospholipid synthesis. Zinc is an essential nutrient required for the growth and metabolism of S. cerevisiae, and of higher eukaryotic cells. The work proposed in this application will address mechanisms by which the expression of key Dhospholipid biosynthetic enzyme activities is regulated in response to zinc depletion. We will examine the lypothesis that zinc depletion induces a cytosolic Mg2+-dependent and NEM-sensitive phosphatidate phosphatase that reduces phosphatidate concentration in the ER. This in turn triggers the Opilp-mediated repression of CHO1 (encodes phosphatidylserine synthase) and other UAS|No-containing genes. This question will be addressed using a phosphatase mutant that will be isolated by a reverse genetic approach requiring the purification of the cytosolic Mg2+-dependent phosphatidate phosphatase enzyme. The regulation of expression of this phosphatase gene by zinc depletion will be examined, along with the biochemical properties of the purified enzyme. We will examine the hypothesis that E/C/7-encoded ethanolamine kinase and CK/t-encoded choline kinase are induced in response to zinc depletion. Mechanisms responsible for the zinc-mediated regulation of the EKI1 and CKI1 genes will be examined, and the biological relevance of regulation for each gene will be examined using selective promoter mutations that affect their expression. The biological relevance of the Opilp-mediated repression of CHO1 and the Zaplp- mediated induction of PIS1 (encodes phosphatidylinositol synthase) will be explored with mutants that are unable to down-regulate phosphatidylserine synthase or up-regulate phosphatidylinositol synthase activities, respectively, in response to zinc depletion. We will examine the hypothesis that the function of the high- affinity plasma membrane zinc transporter Zrt1 p is regulated by changes in phospholipid composition brought about by phospholipid synthesis regulation by zinc depletion. Zinc transport function will be addressed with mutants defective in phospholipid synthesis, and with Zrtlp reconstituted into unilamellar vesicles with varying phospholipid composition.