MITOCHONDRIAL CARRIER STRUCTURE AND FUNCTION

线粒体载体结构和功能分析：线粒体 载体是线粒体内膜上交换的蛋白质 胞质和基质之间的代谢物。他们是中国的关键人物 呼吸、三氯乙酸循环、尿素循环和脂肪酸代谢。 目前基因组测序项目的结果表明，有近 酵母中有50名携带者，线虫中有近200名携带者。因此，它们形成了一个 中等大小的蛋白质家族，对真核生物至关重要。这个 ADP/ATP运输船是这方面研究最深入的成员之一 一群人。它是航母超家族的原型。酵母菌AAC2， 是分析载体结构和功能的理想模型系统。 在基因敲除菌株中表达的突变体可以被纯化和重组 为了测量传输参数，以及强大的遗传方法，如 可以对回复突变进行选择。这个系统有 已经被用来鉴定分子中的八个基本残基 和22个第二位点突变。其中四个回复变种是 表示ADP/ATP载体中本不存在的电荷对 在任何生物化学基础上提出。这项提议将扩大这些成果 通过继续搜索预测存在于 ADP/ATP载体，可能还有大多数其他线粒体载体。在……里面 与ADP/ATP载体的原型角色保持一致，突变体已经 将被用来解决关于这些问题的一个重大悬而未决的问题 承运人。生物物理研究表明，ADP/ATP载体和其他 携带者是二聚体。每个亚基包含六个跨膜片段，但 没有人知道易位途径是否形成于二聚体。 接口，或在每个亚基内独立存在，酵母的遗传学可以 通过表达两个无功能的AAC2突变体来回答这个问题 在基因敲除宿主中分离出质粒。我们已经知道，当D149S 和R252T在单个质粒上表达，它们相辅相成 并产生一种功能蛋白质。如果它们相辅相成 分离蛋白质，那么转位途径必须在二聚体上形成 接口，而不是在两个单个子单元内。这个问题将是 回答。随着一些更大规模的问题得到解决，ADP/ATP 酵母中的载体将允许进行更高分辨率的分析。通过设计一种 不含半胱氨酸的AAC2蛋白，就有可能绘制出螺旋 单体的六个跨膜链段与 同样有趣的是二聚体螺旋之间的接触。曾经是半胱氨酸 可以获得游离蛋白质，半胱氨酸可以放置在 通过形成二硫键来映射螺旋相互作用的序列。成功 对特定二硫键的工程应该识别哪些螺旋是 在结构中相邻并且哪些螺旋面相互接触。 最后，ADP/ATP突变引起的人类疾病问题 承运商已寻址。似乎很明显，这种蛋白质的轻微突变 会导致线粒体不足，这应该会在 疾病，特别是由于能量不足而导致的疾病 利用，如近视眼。提出了一种基于聚合酶链式反应的筛查方法 对于可能存在线粒体缺陷的患者的这些突变。

Analysis of mitochondrial carrier structure and function: Mitochondrial carriers are proteins of the mitochondrial inner membrane that exchange metabolites between the cytosol and the matrix. They are key players in respiration, the TCA cycle, the urea cycle and fatty acid metabolism. Current results from genome sequencing projects suggest there are nearly 50 carriers in yeast and close to 200 in C. elegans. As such, they form a moderately sized protein family of critical importance for eucaryotes. The ADP/ATP carrier is one of the most thoroughly studied members of this group. It serves as an archetype for the carrier superfamily. Yeast AAC2, is an ideal model system for analysis of carrier structure and function. Mutants expressed in a knockout strain can be purified and reconstituted to measure transport parameters, and powerful genetic methods such as selection for revertant mutations can be performed. This system has already been employed to identify eight essential residues in the molecule and 22 second site revertant mutations. Four of these revertants are indicative of charge pairs in the ADP/ATP carrier that would not have been proposed on any biochemical basis. This proposal will extend these results by continuing the search for charge pairs predicted to exist in the ADP/ATP carrier and probably most other mitochondrial carriers as well. In keeping with the archetype role of the ADP/ATP carrier, mutants already made will be used to resolve a major unanswered question about these carriers. Biophysical studies have shown the ADP/ATP carrier and other carriers are dimers. Each subunit contains six transmembrane segments, but no one knows whether the translocation pathway forms at the dimer interface, or independently within each subunit The genetics of yeast can answer this question by expressing two non-functional AAC2 mutants on separate plasmids in the knockout host. We already know that when D149S and R252T are expressed on a single plasmid they complement one another and produce a functional protein. If they complement one another in separate proteins, then the translocation pathway must form at the dimer interface, and not within two single subunits. This question will be answered. As some of the larger scale questions become solved, the ADP/ATP carrier in yeast will permit higher resolution analysis. By engineering a cysteine free AAC2 protein, it will be possible to map the helical contacts between the six transmembrane segments of the monomer and the equally interesting contacts between helices of the dimer. Once a cysteine free protein is available, cysteines can be placed anywhere in the sequence to map helix interactions by disulfide bond formation. Successful engineering of specific disulfide bonds should identify which helices are adjacent in the structure and which helical surfaces are in contact. Finally, the matter of human disease caused by mutations in the ADP/ATP carrier is addressed. It seems clear that mild mutations of this protein would lead to mitochondrial insufficiency that should manifest itself in disease, specifically diseases that are the consequence of poor energy utilization such as myopathies. A PCR based screen is proposed to search for these mutations in patients with possible mitochondrial defects.