Phospholipids and Mitochondrial Function

描述（由申请人提供）： 该建议的中心假设是磷脂酰甘油（PG）和心磷脂（CL）在线粒体功能中发挥确定和特定的作用。已经制备了缺乏CL（crd 1 δ缺乏CL合酶）或PG和CL（pgs 1 δ缺乏PG-磷酸合酶）的酿酒酵母的突变体。这两种突变体在可发酵碳源上生长良好，但前者生长较差，后者在不可发酵碳源上不生长。缺乏PG和CL的细胞的线粒体由于细胞色素c氧化酶（复合物IV）的一个核编码（COX 4）和三个mtDNA编码（COX 1 -3）亚基的翻译缺陷而不能进行氧依赖性能量产生。复合物III的mtDNA编码的细胞色素B（CO B）亚基（细胞色素bc 1）也不被翻译。缺乏CL或PG和CL的酵母活菌株将被用作“生物试剂”，以确定这些阴离子磷脂在正常线粒体功能中的需求的分子基础，从而揭示脂质在细胞功能中的新的和新颖的作用。Specific Aim 1将利用与Cox 4p起始密码子的转录但非翻译结构域5'融合的嵌合报告基因来鉴定负责抑制细胞质中COX 4 mRNA翻译的顺式和反式作用元件。将使用缺失分析、蛋白质- RNA杂交和通过质粒文库互补或诱变抑制翻译缺乏的基因的鉴定。在特定目标2中，将使用类似的方法来鉴定抑制PG/CL缺乏细胞的mtDNA表达的嵌合报告基因的mRNA翻译的顺式和反式作用元件。具体目标3将检验线粒体内膜的能量转导机器对CL具有特定需求的假设。络合物III和IV特异性地结合CL，并且它们形成假定将电子隧道化至分子氧的超分子络合物。组织，稳定性和功能，这个较大的复杂的将被研究作为CL水平和生长条件的函数。复合物III将从crd 1delta细胞中纯化，并在重构的蛋白脂质体中进行研究，以确定CL在其结构和功能中的作用。确定这些脂质在正常线粒体功能中的作用将揭示这些脂质减少的疾病中细胞功能障碍的分子基础。

DESCRIPTION (provided by applicant): The central hypothesis of this proposal is that phosphatidylglycerol (PG) and cardiolipin (CL) play defined and specific roles in the function of the mitochondria. Mutants of the yeast Saccharomyces cerevisiae lacking CL (crd1delta lacking CL synthase) or PG and CL (pgs1delta lacking PG-phosphate synthase) have been made. Both mutants grow well on fermentable carbon sources, but the former grows poorly and the latter does not grow on non-fermentable carbon sources. Mitochondria of cells lacking PG and CL are unable to carry out oxygen-dependent energy production due to a defect in translation of one nuclear encoded (COX4) and three mtDNA encoded (COX1-3) subunits of cytochrome c oxidase (Complex IV). The mtDNA-encoded cytochrome b (COB) subunit of Complex lll (cytochrome bc1) is also not translated. Viable strains of yeast lacking either CL or PG and CL will be used as "biological reagents" to determine the molecular basis for the requirement of these anionic phospholipids in normal mitochondrial function and thereby uncover new and novel roles for lipids in cell function. Specific Aim 1 will utilize chimeric reporter genes fused to the transcribed but untranslated domains 5' of the Cox4p start codon to identify the cis- and trans-acting elements responsible for inhibition of translation of COX4 mRNA in the cytoplasm. Deletion analysis, protein- RNA hybridization, and identification of genes that suppress the lack of translation by plasmid library complementation or mutagenesis will be used. In Specific Aim 2 a similar approach will be used to identify cis- and trans-acting elements that repress translation of the mRNA of chimeric reporter genes expressed by mtDNA of PG/CL-lacking cells. Specific Aim 3 will test the hypothesis that the energy transducing machines of the inner mitochondrial membrane have a specific requirement for CL. Complexes III and IV have specifically bound CL, and they form a supramolecular complex postulated to tunnel electrons to molecular oxygen. The organization, stability, and function of this larger complex will be studied as a function of CL levels and growth conditions. Complex III will be purified from crd1delta cells and studied in reconstituted proteoliposomes to determine the role CL plays in its structure and function. Defining the role these lipids play in normal mitochondrial function will shed light on the molecular basis for cellular dysfunction in diseases where these lipids are reduced.