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，我们开发了通过克隆L合成酶基因和产生零突变体来遗传操纵线粒体特异性磷脂(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。本实验室首次发表了编码CL合成酶的CRD1基因(以前称为CLS1)的克隆，并对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合成酶进行了纯化。我们构建了一个crd1零突变体，它的膜上没有检测到CL。该突变株可以在30摄氏度的可发酵碳源和非发酵碳源上生长，但不能在37摄氏度的碳源上生长。由于缺乏CL和条件致死性，该突变体是进行CL功能体内研究的有力工具。在这个提案中，我们试图了解CL的功能，以及CL合成是如何受到调控的。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扮演什么角色？我们将使用分离crd1缺失突变体的温度敏感性表型的抑制因子的遗传方法，并对抑制基因进行特征分析，以了解为什么CRD1在高温下是必不可少的。此外，我们还将比较crd1缺失突变体和同基因野生型在氧化磷酸化、线粒体膜电位和线粒体通透性转换孔功能方面的差异。2.CL结构基因的表达是如何调控的？我们将使用Northern印迹分析和融合报告基因来确定Pgs1的表达是如何调控的。此外，我们将确定Pgs1p或Crd1p是否受翻译后控制。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来控制结构基因的表达。3.线粒体膜上CL合成酶的活性是如何调节的？我们已经证明CL合成酶是线粒体膜上一个大的复合体的一部分，我们将使用酵母双杂交筛选和化学交联法来鉴定与L合成酶相互作用的复合体的成分。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，占心脏磷脂总量的15%。由于CL对线粒体功能的许多方面都至关重要，因此拟议中的实验结果将为了解心脏的功能提供关键信息。

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.