Functional Analysis of the Oxa1p Export Machinery of Yeast Mitochondria

线粒体是真核细胞中膜分隔的细胞器，在其中发生了许多关键的代谢过程，包括通过氧化磷酸化的复杂过程从营养物质的氧化分解中产生化学能。线粒体实际上有两层膜，将其内部内容物与细胞质隔开。虽然线粒体确实有自己的基因组和自己的蛋白质翻译机制，但线粒体的大多数蛋白质是在核基因组中编码的，并在细胞质中进行生物合成；这些蛋白质随后跨膜转移到线粒体中的适当位置(S)。在特定情况下，位于线粒体内膜上或内的蛋白质(显示出广泛的拓扑排列)，大多数是通过位于外膜和内膜的蛋白质转位机制(分别是TOM和TIM复合体)输入到线粒体中的。一些多位蛋白，呼吸链复合体的所有亚基，由线粒体基因组编码，并在基质间合成。存在许多不同的分选机制，将编码的核蛋白定向到内膜。这些蛋白质的一个子集遵循一个迂回的路线，在这个路线中，它们首先被输入到线粒体基质中，然后走上一条通往细胞膜的“出口”途径。线粒体基因产物也被证明经历了类似的输出机制，以便插入内膜。从线粒体基质到内膜的蛋白质输出与细菌中SEC不依赖的蛋白质输出有相似之处。这些相似之处包括膜电位要求和遵守“正-内”规则，这是前面描述的细菌膜蛋白的分类。最近，在酵母中，核蛋白和线粒体编码蛋白的至少N末端的输出被证明需要内膜蛋白Oxa1p的功能。Oxa1p是酵母线粒体中一种新的蛋白质输出机制的组成部分，它在出口过程中与底物蛋白发生物理作用。Oxa1p在整个进化过程中从原核生物到真核生物(在线粒体和叶绿体中发现)都是保守的。到目前为止，这些非线粒体同源物的功能尚不清楚。人们很容易推测，它们可能在线粒体中发挥与Oxa1p类似的功能，并在细菌中介导跨越叶绿体类囊体膜和跨质膜的蛋白质输出事件(至少是N末端的尾巴)。该项目的主要焦点将是进一步阐明线粒体Oxa1p复合体的功能和组成。将进行的实验的具体目标是：1.确定Oxa1p复合体的功能是否仅限于蛋白质的N末端输出。将分析Oxa1p在相邻跨膜蛋白质片段之间的C-末端尾巴或亲水环输出中的参与。鉴定其他可能与Oxa1p.3在物理或功能上相互作用的蛋白质。确定Oxa1p是否通过卷曲结构形成同源二聚体。探讨Tim17-23输入机制在依赖Oxa1p的蛋白质输出过程中的可能参与。

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.