Molecular Studies of a Mitochondrial Channel

New York University, New York NY

Investigators

Abstract

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).