Functional Analysis of the Oxa1p Export Machinery of Yeast Mitochondria

Marquette University, Milwaukee WI

Investigators

Abstract

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.