PI (H+) AND HOMOLOGOUS MITOCHONDRIAL ANION TRANSPORTERS

线粒体磷酸转运蛋白(PTP)负责 大部分无机磷(PI)在体内的运输 线粒体膜。这种运输(磷酸盐-氢共运输)是 在进行稳态氧化磷酸化时必不可少的 通过细胞中的线粒体：ADP在细胞内被磷酸化为ATP 线粒体基质，而大部分的ATP被用于额外的 线粒体空间，释放PI，它必须被运输回 线粒体。我们已经鉴定了这个蛋白质，重组了运输 高纯度蛋白的活性，测定了其47个N-末端的氨基酸序列 酸，并纯化了牛心脏PTP的cDNA克隆。我们有 发现线粒体之间有显著的序列同源性 ADP/ATP载体和PTP。我们计划获得完整的cdna衍生的氨基 牛心脏组织和大鼠肝脏PTP的酸序列及PTP 来自酿酒酵母的基因。同源区域是 预计将反映进化上的保守和催化上的基本 蛋白质的片段。我们已经确定了一种光亲和性标签 特异性作用于暴露在线粒体基质上的PTP活性部位。 我们计划确定它与之反应的蛋白质片段。其他试剂 将用于探测活动站点(S)和 线粒体膜。我们计划使用定点突变技术 酿酒酵母对催化必需氨基酸的鉴定 PTP的残基或序列片段。我们还计划净化和排序 哺乳动物的ptp基因，以识别组织特异性控制 序列，并与ADP/ATP载体基因一起决定 有一个线粒体转运蛋白多基因家族的成员 具有相似的控制序列，即在 5‘-侧翼，5’-非翻译区，中间和3‘-非翻译区。

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.