Phospholipids and Mitochondrial Function

磷脂和线粒体功能

• 批准号: 7410298
• 负责人: WILLIAM DOWHAN
• 金额: $ 12.74万
• 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/7410298
• 关键词: Binding; Biochemical; Biological; Carbon; Cardiolipins; Cell physiology; Cells; Complex; Condition; Cytochromes; Cytochromes b; Cytoplasm; Defect; Disease; Electron Transport; Electron Transport Complex III; Electrons; Equilibrium; Eukaryotic Cell; Failure; Functional disorder; Genes; Genetic Translation; Genomics; Growth; In Vitro; Initiator Codon; Inner mitochondrial membrane; Libraries; Light; Lipids; Messenger RNA; Mitochondria; Mitochondrial DNA; Molecular; Molecular Genetics; Mutagenesis; Nuclear; Oxygen; Phenotype; Phosphatidyl glycerol; Phosphatidylglycerols; Phospholipids; Plasmids; Play; Production; Proteins; Protons; RNA; Reagent; Regulator Genes; Reporter Genes; Role; Saccharomyces cerevisiae; Source; Structure; Supporting Cell; System; Temperature; Testing; Translating; Translations; Yeasts; base; cDNA Library; cardiolipin synthase; cell growth; cytochrome c oxidase; deletion analysis; dimer; genetic element; in vivo; mitochondrial dysfunction; monomer; mutant; novel; phosphatidylglycerophosphate; proteoliposomes; reconstitution

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 (crdlA lacking CL synthase) or PG and CL (pgslA 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 (CQX4) and three mtDNA encoded (COX1-3) subunits of cytochrome c oxidase (Complex IV). The mtDNA-encoded cytochrome b (COB) subunit of Complex III (cytochrome bcl) 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 Ill 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 crdlA 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.

该提议的中心假设是磷脂酰甘油（PG）和心磷脂（CL）播放定义 以及线粒体功能中的特定作用。酿酒酵母的突变体 缺乏Cl（缺乏CL合酶的CRDLA）或PG和Cl（缺乏PG磷酸PGSLA）已是 制成。两个突变体在可发酵的碳源上生长良好，但前者生长较差，后者生长 不会在不可发酵的碳源上生长。缺乏PG和CL的线粒体无法 由于一个核编码的不足，进行氧依赖的能量产生（CQX4） 和三个MTDNA编码的细胞色素C氧化酶（复合物IV）的亚基（COX1-3）。 mtDNA编码 复合物III（细胞色素BCl）的细胞色素B（COB）亚基也未翻译。可行的酵母菌菌株 缺乏CL或PG和CL将用作“生物试剂”，以确定该分子基础 这些阴离子磷脂在正常的线粒体功能中的需求，从而发现了新的 脂质在细胞功能中的新作用。特定目标1将利用融合的嵌合报道基因 COX4P启动密码子的转录但未翻译的域5'，以识别顺式和反式作用 负责抑制细胞质中Cox4 mRNA翻译的元素。缺失分析，蛋白质 - RNA杂交以及抑制质粒缺乏翻译的基因的鉴定 将使用互补或诱变。在特定目标2中，将使用类似的方法来识别 顺式和反式作用元素，这些元素抑制嵌合报道基因mRNA的翻译。 pg/cl倾斜细胞的mtDNA。特定的目标3将检验能量传递机器的假设 内部线粒体膜的特定要求。复合物生病和IV有 特定结合的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到分子 氧。该较大复合物的组织，稳定性和功能将作为CL的函数研究 水平和生长条件。复合物III将从CRDLA单元中纯化并在重构中进行研究 蛋白脂质体确定Cl在其结构和功能中的作用。定义这些脂质的角色 在正常的线粒体功能中发挥作用将揭示出分子基础的细胞功能障碍 这些脂质减少的疾病。