Mitochondrial Protein Import: a Mutagenesis Approach to Studying the Structural and Functional Properties of Tim50

The mitochondrion is an essential organelle functioning in various pathways in the eukaryotic cell from energy production, metabolism of amino acids and lipids, assembly of iron sulphur clusters, to the regulation of apoptosis. Throughout evolution mitochondrial genes were shifted to the nuclear genome. Therefore, despite the fact that the mitochondrion contains its own circular DNA and ribosomes, most (99%) mitochondrial proteins are encoded by the nucleus. These proteins are translated on cytosolic ribosomes and are sorted via large complexes serving as translocases and insertases to one of the four mitochondrial compartments: outer membrane, inner membrane, intermembrane space and the matrix.

Our research focuses on the translocase of the inner membrane complex (TIM23) that mediates the import of proteins targeted to the matrix and some of the inner membrane and IMS proteins. Tim50 is an essential subunit of this complex which serves as a receptor that recognizes the precursor and transfers it to Tim23 which forms the import channel. Both proteins expose conserved domains into the IMS that interact with each other. This Tim23-Tim50 interaction is essential for proper protein import across the mitochondrial inner membrane.

The objective of this work is to elucidate the molecular mechanism of function of the yeast TIM23 complex with emphasis on the role of Tim50. For this purpose we will create a large set of Tim50 mutants in order to screen for mutations that may cause temperature sensitive growth in yeast. The isolated mutants will be sequenced and the character of their impaired interaction will be determined.