Phospholipids and Mitochondrial Function

这一建议的中心假设是磷脂酰甘油(PG)和心磷脂(CL)发挥作用 以及在线粒体功能中的特定作用。酿酒酵母的突变株 缺乏CL(crdlA缺乏CL合成酶)或PG和CL(pgslA缺乏PG-磷酸合成酶) 制造。这两个突变株在可发酵的碳源上生长良好，但前者生长较差，后者生长较差。 不能在非发酵的碳源上生长。缺乏PG和CL的细胞线粒体不能 由于一个核编码(CQX4)的翻译缺陷而进行氧气依赖的能量生产 3个线粒体DNA编码细胞色素C氧化酶亚基(COX1-3)(复合体IV)。线粒体DNA编码 复合体III的细胞色素b(COB)亚单位(细胞色素BCL)也不被翻译。有活力的酵母菌株 缺少CL或PG，而CL将被用作“生物试剂”，以确定 这些阴离子磷脂对正常线粒体功能的需求，从而发现新的和 脂质在细胞功能中的新作用。特定目标1将利用嵌合报告基因 Cox4p起始密码子转录但未翻译的结构域5‘以识别顺式和反式作用 负责抑制胞浆中COX4 mRNA翻译的元件。缺失分析，蛋白质- RNA杂交，利用质粒库鉴定抑制翻译缺失的基因 将使用互补或诱变。在具体目标2中，将使用类似的办法来确定 抑制嵌合报告基因mRNA翻译的顺式和反式作用元件 PG/CL缺失细胞的线粒体DNA。具体目标3将检验这样一种假设，即能量转换机器 线粒体膜的内层对CL有特殊的要求。复合体Ill和IV有 它们特别地结合了CL，形成了一种超分子络合物，假定它可以将电子隧道到分子中 氧气。将作为CL的函数来研究这个较大综合体的组织、稳定性和功能 水平和生长条件。将从crdlA细胞中纯化复合体III，并在重组的 蛋白质脂质体以确定CL在其结构和功能中所起的作用。定义这些脂质的作用 正常线粒体功能的发挥将揭示细胞功能障碍的分子基础 这些脂类减少的疾病。

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.