Phospholipids and Mitochondrial Function

描述(由申请人提供)： 这一建议的中心假设是磷脂酰甘油(PG)和心磷脂(CL)在线粒体的功能中发挥明确和特定的作用。对缺乏CL(crd1Delta缺失CL合成酶)或pg和CL(pgs1Delta缺失PG-磷酸合成酶)的酿酒酵母进行了突变。这两个突变株在可发酵碳源上生长良好，但前者生长不良，后者不能在非发酵碳源上生长。缺乏PG和CL的细胞线粒体不能进行氧依赖的能量产生，这是由于细胞色素C氧化酶(复合体IV)的一个核编码(COX4)亚基和三个mtDNA编码(COX1-3)亚基的翻译缺陷。线粒体DNA编码的细胞色素b(COB)亚单位的复合体111(细胞色素Bc1)也没有被翻译。缺乏CL或PG和CL的活性酵母菌株将被用作“生物试剂”，以确定这些阴离子磷脂在正常线粒体功能中所需的分子基础，从而揭示脂类在细胞功能中的新的和新的作用。特异性目标1将利用融合到Cox4p起始密码子转录但未翻译区域5‘的嵌合报告基因来识别负责抑制细胞质中COX4 mRNA翻译的顺式和反式作用元件。将使用缺失分析、蛋白质-RNA杂交和鉴定通过质粒库互补或突变抑制翻译缺失的基因。在特定目标2中，将使用类似的方法来鉴定抑制PG/CL缺失细胞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将检验线粒体膜内的能量转换机对CL有特殊要求的假设。络合物III和IV与CL有特异的结合，它们形成了一种超分子络合物，假定它能将电子隧道传输到分子氧。这个更大的复合体的组织、稳定性和功能将作为CL水平和生长条件的函数进行研究。复合体III将从crd1Delta细胞中提纯，并在重组蛋白脂质体中进行研究，以确定CL在其结构和功能中所起的作用。确定这些脂质在正常线粒体功能中所起的作用，将有助于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.