Molecular Studies of a Mitochondrial Channel

蛋白质跨膜转运是原核生物和真核生物的重要过程。 蛋白质易位对多种细胞功能至关重要，包括蛋白质分泌、细胞器的生物发生、区室化和细胞信号传导。由于在细胞中合成的蛋白质中约有一半在到达其最终目的地之前必须穿过至少一个膜，因此蛋白质跨膜转运是一个基本的细胞过程。前体蛋白质被输入到双膜细胞器的过程不仅本身很重要，而且它也是研究这一普遍存在的过程的模型系统。在线粒体的内膜和外膜中存在复杂的蛋白质组，其识别、分选和选择性地将前体蛋白质跨各自的膜转运到其最终目的地。外膜输入机制被称为汤姆（易位，外膜）复合体，内膜机制被称为蒂姆（易位，内膜）复合体。复合物的组分被指定为Tim或Tom，随后是它们的分子量。完整的膜蛋白由Tim 22复合物输入。充水通道据称在这一过程中发挥关键作用，在内膜和外膜，这些通道被命名为TIM和TOM，分别。该项目的一个主要目标是使用酵母线粒体来定义这些线粒体通道在蛋白质输入过程中的作用和调节机制。这些发现应该适用于其他细胞膜和高等真核生物的这一过程。天然线粒体内膜和外膜的蛋白质输入通道将通过使用称为“膜片钳”的技术进行研究，其中在一小片膜中的单个通道上进行电生理测量。 来自各种基因操作的酵母菌株的线粒体和线粒体体（通过French压榨剥离其外膜的线粒体）将进行直接膜片钳。将记录电生理通道活动，并与使用重组Tim蛋白在蛋白质易位中发挥主要作用的蛋白质脂质体中重建的线粒体通道活动进行比较。特异性重组Tim蛋白（例如，23、17和50）将与在有丝分裂体和蛋白脂质体中发现的TIM进行比较。本文还将研究TOM的单通道特性，包括其通过靶向肽的调节和Tom复合物的结构。在建立通道激活方案后，将重组Tim 22的通道活性与在线粒体中发现的通道活性进行比较。蛋白质通过TIM和TOM转运的机制将在实际蛋白质输入过程中通过膜片钳遗传操纵的线粒体和有丝分裂体进一步研究。复合物的结构和前蛋白折叠对进口的影响将使用突变体和模型蛋白来解决。作为美国唯一一家拥有将这些技术常规应用于天然和重组线粒体膜的专业知识的实验室的负责人，Kinnally博士处于独特的位置，可以对线粒体中蛋白质输入的机制进行研究。虽然这些实验将使研究线粒体中蛋白质易位的机制和通道的作用成为可能，但Kinnally博士的研究成果将有助于研究线粒体中蛋白质转运的机制和通道的作用。很可能在所有生物膜中遵循相同的原理。因此，理解线粒体分选和蛋白质易位可以促进理解，例如，在原核生物，内质网和叶绿体中的相同过程。该项目的结果将被公布，以增加基本细胞过程的信息基础，即，蛋白质跨膜转运。这些信息是这一领域和其他领域的研究以及教育的资源。PI对科学人力资源的开发非常感兴趣。许多研究生、本科生和高中生、博士后、资深科学家和教师都曾访问过该实验室并/或在该实验室接受过培训（这种培训具有额外的好处，可以作为传播科学成果和实验室独特技术的进一步机制）。

Protein translocation across membrane barriers is an important process in all organisms, both prokaryotic and eukaryotic. Protein translocation is critical to a variety of cellular functions, including protein secretion, biogenesis of organelles, compartmentalization, and cell signalling. Since about half of the proteins synthesized in a cell must cross at least one membrane before reaching their final destinations, protein translocation across membranes is a fundamental cellular process. The process by which precursor proteins are imported into the mitochondrion, a double-membraned organelle, is not only important in its own right, but it also serves as a model system for studying this ubiquitous process. There are complex sets of proteins in both the inner and outer membranes of mitochondria that recognize, sort and selectively translocate precursor proteins across each respective membrane to their final destinations. The outer membrane import machinery is called the Tom (translocation, outer membrane) complex and the inner membrane machinery is called the Tim (translocation, inner membrane) complex. Components of the complexes are designated Tim or Tom followed by their molecular weight. Integral membrane proteins are imported by the Tim22 complex. Water-filled channels are purported to play key roles in this process in both the inner and outer membranes, and these channels are named TIM and TOM, respectively. A major goal of this project is to define the mechanism of action and regulation of these mitochondrial channels during protein import, using yeast mitochondria. The findings should be applicable to this process in other cell membranes and higher eukaryotes. The protein import channels of the native mitochondrial inner and outer membranes will be studied by using a technique called "patch-clamping" in which electrophysiological measurements are made across single channels in a small patch of membrane. Mitochondria and mitoplasts (mitochondria stripped of their outer membrane by French pressing) from various genetically manipulated strains of yeast will be subjected to direct patch-clamping. The electrophysiological channel activities will be recorded and compared with those of mitochondrial channels reconstituted in proteoliposomes using recombinant Tim proteins thought to play a major role in protein translocation. The single channel behavior and peptide sensitivity of specific recombinant Tim proteins (e.g., 23, 17 and 50) will be compared with TIM found in mitoplasts and proteoliposomes. The the single channel properties of TOM will also be studied, including its regulation by targeting peptides and architecture of the Tom complex. The channel activity of recombinant Tim22 will be compared with that found in the mitoplast, after establishing channel activation protocols. The mechanism of protein translocation through TIM and TOM will be further examined by patch-clamping genetically manipulated mitochondria and mitoplasts during actual protein import. The architecture of the complexes and the effects of preprotein folding on import will be addressed using mutants and model proteins. As head of the only lab in the U.S. with the expertise to routinely apply these techniques to native and reconstituted mitochondrial membranes, Dr. Kinnally is in a unique position to undertake these investigations of the mechanisms of protein import in mitochondria.While these experiments will enable studies of the mechanism(s) and the role of channels in protein translocation in mitochondria, it is likely that the same principles are followed in all biological membranes. Therefore, understanding mitochondrial sorting and protein translocation can facilitate understanding of, for example, the same process in the prokaryotes, endoplasmic reticulum and chloroplasts. The results from this project will be published to increase the information base of a fundamental cellular process, i.e., protein translocation across membranes. This information is a resource for research in this and other areas, as well as education. The PI is keenly interested in development of human resources in science. Many graduate, undergraduate, and high school students, postdoctorals, senior scientists and teachers have visited and/or trained in this laboratory (this training has the added benefit of serving as a further mechanism for dissemination of both scientific results and the techniques uniquely employed in the laboratory).