Phospholipids and Mitochondrial Function

磷脂和线粒体功能

• 批准号: 6734690
• 负责人: WILLIAM DOWHAN
• 金额: $ 37.79万
• 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 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6734690
• 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了定义和特定作用。缺乏Cl（缺乏CL合酶的CRD1DELTA）或PG和Cl（PGS1DELTA缺乏PG-磷酸合酶）的酵母菌的突变体。这两个突变体在可发酵碳源上生长良好，但前者的生长较差，而后者并不能在不可发酵的碳源上生长。缺乏PG和CL的线粒体由于在一个核编码（COX4）和三个MTDNA编码（COX1-3）亚基的核C氧化酶（复杂IV）的核能转换和三个MTDNA（COX1-3）亚基，因此无法进行氧依赖性能量产生。 MTDNA编码的复合LLL（细胞色素BC1）的细胞色素B（COB）亚基也未翻译。缺乏CL或PG和CL的可行酵母菌菌株将用作“生物试剂”，以确定这些阴离子磷脂在正常的线粒体功能中需要的分子基础，从而发现细胞功能中脂质的新作用。具体目标1将利用与Cox4p启动密码子5'conscropted但未翻译的域5'融合的嵌合报道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。缺失分析，蛋白质杂交和鉴定抑制质粒文库互补或诱变缺乏翻译的基因。在特定的目标2中，将使用类似的方法来识别顺式和反式作用元件，这些元素抑制了由PG/Cl-lack-lacking细胞mtDNA表达的嵌合报告基因mRNA的翻译。特定的目标3将检验以下假设：内部线粒体膜的能量转导机具有对CL的特定要求。复合物III和IV具有特异性结合的Cl，它们形成了一个超分子复合物，该复合物假定为隧道电子与分子氧。该较大复合物的组织，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，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。定义这些脂质在正常线粒体功能中的作用将揭示出降低这些脂质的疾病中细胞功能障碍的分子基础。