CARDIOLIPIN BIOSYNTHESIS AND FUNCTION

我们的长期目标是了解磷脂在线粒体膜的组装，组织和功能中的作用。为此，我们已经开发了通过克隆编码L合酶的基因和产生无效突变体来遗传操纵心磷脂（CL）水平的工具。先前的体外研究表明，CL对几种线粒体酶的功能至关重要。然而，到目前为止，还不可能扩展这些信号来揭示CL在体内的作用。我们现在有了这样做的分子工具。本实验室首次克隆了编码CL合成酶的CRD 1基因（以前称为CLS 1），并纯化了CL途径的两个关键酶，磷脂酰甘油磷酸合成酶（PGS1基因产物）和CL合成酶。我们构建了一个crd 1无效突变体，其膜中没有可检测到的CL。crd1突变体可以在30摄氏度下在可发酵和不可发酵碳源上生长，但不能在37摄氏度下生长。由于其缺乏CL和条件致死性，突变体是进行CL功能体内研究的有力工具。在这个提议中，我们试图了解CL的功能，以及CL的合成是如何调节的。拟议的实验将解决以下问题：1。CL在线粒体功能和细胞活力中的作用是什么？我们将使用的遗传方法分离抑制剂的温度敏感性表型的crd1无效突变体，并表征抑制基因，以了解为什么CRD1是必不可少的在高温下。此外，我们将比较crd 1无效突变体和同基因野生型的氧化磷酸化，线粒体膜电位，线粒体通透性转换孔的功能。2. CL结构基因的表达是如何调节的？我们将使用北方印迹分析和融合报告基因，以确定如何PGS1表达的调控。此外，我们将确定Pgs1p或Crd1p是否在术后得到控制。最后，我们将确定影响CL合成的调控基因，并确定它们如何相互作用以控制结构基因3的表达。CL合酶活性在线粒体膜中是如何调节的？我们已经表明，CL合酶是一个大的复合物在线粒体膜的一部分，我们将使用酵母双杂交筛选和化学交联，以确定与L合酶相互作用的复合物的组件。然后我们将确定这些成分的缺失或过度表达如何影响CL合酶活性。CL是哺乳动物心脏中主要的聚甘油磷脂，占总心脏磷脂质量的15%。由于CL对线粒体功能的许多方面都至关重要，因此拟议实验的结果将为了解心脏功能提供关键信息。

Our long range goal is to understand the role of phospholipids in the assembly, organization, and function of the mitochondrial membrane. Toward this end, we have developed the tools to genetically manipulate the levels of the mitochondria-specific phospholipid cardiolipin (CL) through cloning of the gene encoding L synthase and generation of a null mutant. Previous in vitro studies had suggested that CL was critical to the function of several mitochondrial enzymes. However, until now, it has been impossible to extend these signals to reveal a role for CL in vivo. We now have the molecular tools to do so. Our lab was the first to publish the cloning of the CRD1 gene (previously called CLS1) encoding CL synthase, and the purification of the two key enzymes of the CL pathway, phosphatidylglycerolphosphate synthase (the PGS1 gene product) and CL synthase. We constructed a crd1 null mutant which has no detectable CL in its membranes. The crd1 mutant can grow on both fermentable and non-fermentable carbon sources at 30 degrees Centigrade, but cannot grow at 37 degrees Centigrade. With its lack of CL and conditional lethality, the mutant is a powerful tool with which to carry out in vivo studies of CL function. In this proposal, we seek to understand the function of CL, and how CL synthesis is regulated. Proposed experiments will address the following questions: 1. What is the role of CL in mitochondrial function and cell viability? We will use the genetic approach of isolating suppressors of the temperature sensitivity phenotype of the crd1 null mutant, and characterizing the suppressor genes to understand why CRD1 is essential at elevated temperatures. In addition, we will compare the crd1 null mutant and isogenic wild type with respect to oxidative phosphorylation, mitochondrial membrane potential, and function of the mitochondrial permeability transition pore. 2. How is expression of the CL structural genes regulated? We will use northern blot analysis and fusion to reporter genes to determine how PGS1 expression is regulated. In addition, we will determine if Pgs1p or Crd1p are controlled post-translationally. Finally, we will identify regulatory genes which affect CL synthesis and determine how they interact to control expression of the structural genes 3. How is CL synthase activity regulated in the mitochondrial membrane? We have shown that CL synthase is part of a large complex in the mitochondrial membrane We will use both the yeast two-hybrid screen and chemical cross-linking to identify components of the complex which interact with L synthase. We will then determine how deletion or over- expression of these components affects CL synthase activity. CL is the major polyglycerolphospholipid in the mammalian heart, comprising 15% of the total cardiac phospholipid mass. Because CL is crucial for many aspects of mitochondrial function, the results of the proposed experiments will provide information critical to understanding how the heart functions.