MITOCHONDRIAL CARRIER STRUCTURE AND FUNCTION

线粒体载体结构和功能分析：线粒体 载体是线粒体内膜的蛋白质， 细胞质和基质之间的代谢物。他们是关键参与者， 呼吸、TCA循环、尿素循环和脂肪酸代谢。 目前的基因组测序结果表明， 酵母菌中有50个载体，C.优雅因此，它们形成了一个 对真核生物至关重要的中等大小的蛋白质家族。的 ADP/ATP载体是其中研究最透彻的成员之一， 组它作为载体超家族的原型。酵母AAC2， 是分析载体结构和功能的理想模型系统。 在敲除菌株中表达的突变体可以被纯化和重构 测量传输参数，以及强大的遗传方法， 可以进行回复突变体的选择。该系统具有 已经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体。其中四种回复突变体是 指示ADP/ATP载体中的电荷对， 任何生物化学的基础上提出的。该提案将扩展这些成果 通过继续搜索预测存在于 ADP/ATP载体，可能也是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 将被用来解决一个主要的悬而未决的问题，这些 载波生物物理学研究表明，ADP/ATP载体和其他 载体是二聚体。每个亚基含有六个跨膜片段，但 没有人知道易位途径是否在二聚体上形成 界面，或独立地在每个亚基内酵母的遗传学可以 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 并产生一种功能性蛋白质。如果他们互相补充， 分离的蛋白质，那么易位途径必须在二聚体处形成， 接口，而不是在两个单独的子单元内。该问题将被 回答随着一些大规模问题的解决，ADP/ATP 酵母中的载体将允许更高分辨率的分析。通过工程化 不含半胱氨酸的AAC2蛋白，将有可能绘制螺旋 单体的六个跨膜区段与膜之间的接触 二聚体的螺旋之间的同样有趣的接触。一旦半胱氨酸 游离蛋白质是可用的，半胱氨酸可以放置在任何地方， 序列通过二硫键形成来映射螺旋相互作用。成功 特异性二硫键的工程设计应该确定哪些螺旋是 在结构中相邻并且螺旋表面接触。 最后，由ADP/ATP突变引起的人类疾病 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 疾病，特别是由于能量不足而导致的疾病 利用，如肌病。提出了一种基于PCR的屏幕搜索 在可能有线粒体缺陷的患者中检测这些突变。

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.