Functional Analysis of the Oxa1p Export Machinery of Yeast Mitochondria

线粒体是真核细胞的膜界定的细胞器，其中发生许多关键的代谢过程，包括通过称为氧化磷酸化的复杂过程从营养物质的氧化分解产生化学能。 实际上，细胞膜上有两层膜，将其内部物质与细胞质隔开。 虽然转录子确实有自己的基因组和蛋白质翻译机制，但转录子的大多数蛋白质都在核基因组中编码，并在细胞质中生物合成;这些蛋白质随后穿过膜转移到转录子内的适当位置。 在位于线粒体内膜上或内膜中的蛋白质的特定情况下（并且其显示出广泛的拓扑排列），大多数通过位于外膜和内膜中的蛋白质易位机制（分别为TOM和TIM复合物）输入线粒体。 线粒体基因组编码的一些多位蛋白是呼吸链复合物的亚基，它们在基质区室中合成，并通过多种不同的分选机制将核内编码的蛋白定向到内膜。这些蛋白质的一个子集遵循迂回的路线，其中它们首先被输入到线粒体基质中，然后走上“出口”途径到膜。 线粒体基因产物也被证明经历类似的输出机制，以便插入内膜。从线粒体基质到内膜的蛋白质出口与细菌中的SEC独立蛋白质出口具有相似性。这些相似之处包括膜电位要求和遵守“阳性内部”规则，如前所述的细菌膜蛋白的分选。最近，在酵母中，核和细胞内编码的蛋白质的至少N-末端尾部的输出已被证明需要内膜蛋白Oxa 1 p的功能。Oxa 1 p是酵母细胞中一种新型蛋白质输出机制的组成部分，在原核生物和真核生物的进化过程中（存在于线粒体和叶绿体中）都是保守的。这些非线粒体同源物的功能至今尚不清楚。这是诱人的推测，他们可能执行类似的功能，Oxa 1 p在线粒体和介导蛋白质输出事件（至少N-末端尾巴）跨类囊体膜在叶绿体和跨质膜在细菌。该项目的主要焦点将是进一步阐明线粒体Oxa 1 p复合物的功能和组成。将进行的实验的具体目标是：1。确定Oxa 1 p复合物的功能是否仅限于蛋白质N-末端尾部的输出。将分析Oxa 1 p参与蛋白质跨膜片段之间的C-末端尾或亲水环的输出。鉴定可能与Oxa1p.3发生物理或功能相互作用的其他蛋白质。确定Oxa 1 p是否通过卷曲螺旋结构形成同源二聚体。探讨Tim 17 -23输入机制在Oxa 1 p依赖蛋白输出过程中的可能作用。

The mitochondrion is the membrane-delimited organelle of eukaryotic cells within which a number of key metabolic processes occur, including the generation of chemical energy from the oxidative breakdown of nutrients via the complex process known as oxidative phosphorylation. The mitochondrion actually has two membranes separating its inner content from the cytoplasm of the cell. Although the mitochondrion does have its own genome and its own protein translational machinery, most of the proteins of the mitochondrion are encoded in the nuclear genome and are biosynthesized in the cytoplasm; these proteins are subsequently translocated across the membrane(s) to their appropriate place within the mitochondrion. In the particular case of proteins that sit on or in the inner mitochondrial membrane (and which display a wide range of topological arrangements), most are imported into the mitochondria via protein translocation machineries located in the outer and inner membranes (TOM and TIM complexes, respectively). A few polytopic proteins, all subunits of respiratory chain complexes, are encoded by the mitochondrial genome and are synthesized within the matrix compartment.A number of distinct sorting mechanisms exist to direct nuclearly encoded proteins to the inner membrane. A subset of these proteins follow a circuitous route in which they are first imported into the mitochondrial matrix and then embark on an "export" pathway to the membrane. Mitochondrial gene products have also been shown to undergo a similar export mechanism in order to become inserted into the inner membrane. Protein export from the mitochondrial matrix into the inner membrane bears similarities to Sec-independent protein export in bacteria. These similarities include membrane potential requirements and adherence to the "positive-inside" rule, described previously for the sorting of bacterial membrane proteins. Recently in yeast, the export of at least the N-terminal tails of both nuclear and mitochondrially encoded proteins has been shown to require the function of an inner membrane protein, Oxa1p. Oxa1p, which physically interacts with substrate proteins as they are undergoing export, has been proposed to represent a component of a novel protein export machinery in yeast mitochondria.Oxa1p is conserved throughout evolution, from prokaryotes throughout eukaryotes (where it is found in mitochondria and chloroplasts). The function of these non-mitochondrial homologs is not known to date. It is tempting to speculate that they may perform a similar function as Oxa1p in mitochondria and mediate protein export events (at least of N-terminal tails) across the thylakoid membrane in chloroplasts and across the plasma membrane in bacteria. The major focal point of this project will be the further elucidation of the function and composition of the mitochondrial Oxa1p complex. The specific goals of the experiments that will be performed are:1. To determine if the function of the Oxa1p complex is limited to the export of N-terminal tails of proteins. The involvement of Oxa1p in the export of C-terminal tails or hydrophilic loops between neighboring membrane-spanning segments of proteins will be analyzed.2. To identify other proteins which may physically or functionally interact with Oxa1p.3. To determine whether Oxa1p forms homo-dimers through coiled-coil structures.4. To investigate the possible involvement of the Tim17-23 import machinery in the process of Oxa1p-dependent protein export.