MITOCHONDRIAL CARRIER STRUCTURE AND FUNCTION

线粒体载体结构与功能分析：线粒体 载体是线粒体内膜上进行交换的蛋白质 细胞质和基质之间的代谢物。他们是关键人物 呼吸作用、TCA循环、尿素循环和脂肪酸代谢。 基因组测序项目的当前结果表明，几乎有 酵母中有 50 个携带者，秀丽隐杆线虫中有近 200 个携带者。因此，他们形成了一个 对真核生物至关重要的中等大小的蛋白质家族。的 ADP/ATP 载体是该领域研究最深入的成员之一 组。它是载体超家族的原型。酵母AAC2， 是分析载体结构和功能的理想模型系统。 敲除菌株中表达的突变体可以被纯化和重建 测量运输参数，以及强大的遗传方法，例如 可以进行回复突变的选择。该系统有 已被用来鉴定分子中的八个必需残基 和22个第二位点回复突变。其中四个回复体是 表明 ADP/ATP 载体中的电荷对本来不会 在任何生化基础上提出。该提案将扩展这些结果 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 与 ADP/ATP 载体的原型作用保持一致，突变体已经 made 将用于解决有关这些问题的一个未解答的主要问题 载体。生物物理学研究表明 ADP/ATP 载体和其他 载体是二聚体。每个亚基包含六个跨膜片段，但是 没有人知道易位途径是否在二聚体处形成 酵母的遗传学可以 通过表达两个非功能性 AAC2 突变体来回答这个问题 在敲除宿主中分离质粒。我们已经知道当D149S 和 R252T 在单个质粒上表达，它们彼此互补 并产生功能性蛋白质。如果他们在各方面互补 分离蛋白质，那么易位途径必须在二聚体处形成 接口，而不是在两个单个子单元内。这个问题将是 回答道。随着一些更大规模问题的解决，ADP/ATP 酵母中的载体将允许更高分辨率的分析。通过设计一个 不含半胱氨酸的 AAC2 蛋白，将有可能绘制螺旋图 单体的六个跨膜片段和 二聚体螺旋之间的接触同样有趣。一旦半胱氨酸 可获得游离蛋白质，半胱氨酸可以放置在任何位置 通过二硫键形成来绘制螺旋相互作用的序列。成功 特定二硫键的工程应确定哪些螺旋是 结构中相邻且螺旋表面接触。 最后，ADP/ATP 突变引起的人类疾病问题 承运人已解决。很明显，这种蛋白质的轻微突变 会导致线粒体功能不全，这应该表现在 疾病，特别是由于能量不足而导致的疾病 利用如肌病。建议使用基于 PCR 的筛选来搜索 针对可能存在线粒体缺陷的患者中的这些突变。

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.