Phospholipids and Mitochondrial Function

磷脂和线粒体功能

• 批准号: 7046804
• 负责人: WILLIAM DOWHAN
• 金额: $ 38.66万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7046804
• 关键词: Saccharomyces cerevisiae; auxotrophy; bioenergetics; cardiolipins; cytochrome b; cytochrome c; cytochrome oxidase; enzyme biosynthesis; enzyme complex; enzyme mechanism; fungal genetics; gene deletion mutation; genetic regulation; genetic transcription; genetic translation; mitochondria; mitochondrial DNA; phosphoglycerides; protein purification

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.

描述（由申请人提供）： 该提案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明确且特定的作用。缺乏 CL（缺乏 CL 合酶的 crd1delta）或 PG 和 CL（缺乏 PG-磷酸合酶的 pgs1delta）的酿酒酵母突变体已经被制成。两种突变体在可发酵碳源上生长良好，但前者生长不良，后者在非发酵碳源上不生长。由于细胞色素 c 氧化酶（复合体 IV）的一个核编码 (COX4) 和三个 mtDNA 编码 (COX1-3) 亚基的翻译缺陷，缺乏 PG 和 CL 的细胞线粒体无法进行氧依赖性能量产生。复合体III（细胞色素bc1）的mtDNA编码的细胞色素b（COB）亚基也不被翻译。缺乏 CL 或 PG 和 CL 的酵母活菌株将被用作“生物试剂”，以确定正常线粒体功能中这些阴离子磷脂需求的分子基础，从而揭示脂质在细胞功能中的新的和新颖的作用。具体目标 1 将利用与 Cox4p 起始密码子的转录但非翻译域 5' 融合的嵌合报告基因来鉴定负责抑制细胞质中 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杂交以及通过质粒文库互补或诱变来抑制翻译缺失的基因的鉴定。在具体目标 2 中，将使用类似的方法来鉴定抑制由缺乏 PG/CL 的细胞的 mtDNA 表达的嵌合报告基因 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换机器对 CL 有特定要求的假设。配合物 III 和 IV 特异性结合 CL，并且它们形成超分子配合物，假定将电子隧道传输至分子氧。这个较大复合体的组织、稳定性和功能将作为 CL 水平和生长条件的函数进行研究。复合物 III 将从 crd1delta 细胞中纯化出来，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。定义这些脂质在正常线粒体功能中所起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。