PI (H+) AND HOMOLOGOUS MITOCHONDRIAL ANION TRANSPORTERS

PI (H ) 和同源线粒体阴离子转运蛋白

• 批准号: 3282980
• 负责人: Hartmut none Wohlrab
• 金额: $ 25.4万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282980
• 关键词: Saccharomyces; acidity /alkalinity; adenine nucleotides; affinity labeling; chemical structure function; complementary DNA; gel electrophoresis; genetic manipulation; heart; ion transport; laboratory rabbit; laboratory rat; liver; membrane permeability; mitochondria; mitochondrial membrane; molecular cloning; nuclear magnetic resonance spectroscopy; phosphates; point mutation; protein reconstitution; protein sequence; protein structure; radiotracer; transport proteins

The mitochondrial phosphate transport protein (PTP) is responsible for the transport of most of the inorganic phosphate (Pi) across the inner mitochondrial membrane. This transport (Phosphate-Hydrogen cotransport) is essential for steady state oxidative phosphorylation as it is carried out by mitochondria in the cell: ADP is phosphorylated to ATP in the mitochondrial matrix while most of the ATP is utilized in the extra mitochondrial space, liberating Pi, which must be transported back into the mitochondria. We have identified this protein, reconstituted the transport activity of the highly purified protein, sequenced its 47 N-terminal amino acids, and have purified a bovine cardiac PTP cDNA clone. We have discovered significant sequence homologies between the mitochondrial ADP/ATP carrier and PTP. We plan to obtain the complete cDNA-derived amino acid sequence of PTP from bovine cardiac tissue and rat liver and the PTP gene from the yeast Saccharomyces cerevisiae. Homologous regions are expected to reflect evolutionarily conserved and catalytically essential segments of the protein. We have identified a photoaffinity label that acts specifically on the mitochondrial matrix exposed active site of PTP. We plan to identify the protein segment it reacts with. Other reagents will be used to probe the active site(s) and the topology of PTP in the mitochondrial membrane. We plan to use site-specific mutagenesis in Saccharomyces cerevisiae to identify catalytically essential amino acid residues or sequence segments of PTP. We also plan to purify and sequence the mammalian PTP gene, in order to identify tissue-specific control sequences and, together with the ADP/ATP carrier gene, determine whether there is a mitochondrial transport protein multigene family with members having similar control sequences, i.e. conserved sequence segments in the 5'-flanking, 5'-untranslated, intervening, and 3'-untranslated regions.

线粒体磷酸盐转运蛋白（PTP）负责 大部分无机磷酸盐（Pi）通过内层的运输 线粒体膜 这种运输（磷酸盐-氢协同运输）是 对于进行稳态氧化磷酸化至关重要 在细胞中的线粒体：ADP在细胞中磷酸化为ATP。 线粒体基质，而大部分ATP用于额外的 线粒体空间，释放Pi，它必须被运送回线粒体空间。 线粒体 我们已经鉴定了这种蛋白质， 对高度纯化的蛋白进行活性测定，并对其N-末端47个氨基酸进行序列测定 酸，并纯化了牛心脏PTP cDNA克隆。 我们有 发现了线粒体之间的重要序列同源性 ADP/ATP载体和PTP。 我们计划获得完整的cDNA衍生的氨基 牛心肌和大鼠肝PTP的氨基酸序列及PTP 来自酿酒酵母的基因。 霍姆斯地区是 预计反映了进化上保守和催化必需的 蛋白质的片段。 我们已经确定了一种光亲和标记， 特异性作用于线粒体基质暴露的PTP活性位点。 我们计划确定与之反应的蛋白质片段。 其他试剂 将用于探测活动站点和 线粒体膜 我们计划使用定点突变， 酿酒酵母鉴定催化必需氨基酸 PTP的残基或序列片段。 我们还计划净化和测序 哺乳动物PTP基因，以确定组织特异性控制 序列，并与ADP/ATP载体基因一起，决定是否 线粒体转运蛋白多基因家族 具有相似的控制序列，即在基因组中的保守序列区段， 5 '-侧翼区、5'-非翻译区、间插区和3 '-非翻译区。