Protein translocation across membrane barriers is important in many cellular functions including signaling, secretion, biogenesis of organelles, compartmentalization, and programmed cell death. A much studied yet poorly understood example is the process by which precursor proteins are imported into mitochondria. A complex set of proteins in both the inner and outer membranes of this organelle recognizes, sorts and selectively translocates precursors to their final destinations. A water-filled proteinaceous pore called the multiple conductance channel (MCC) of the inner membrane is postulated to play a key role in this process. This project has three objectives related to its main goal of determining the physiological role of the MCC in protein import and the molecular basis of its action. The hypothesis that the MCC is the import pore predicts that interfering with other components of the import apparatus will change the properties of the MCC. Therefore, treatment with antibodies and proteolysis of the hetero-oligomeric import apparatus of the inner membrane will be used to test for effects on MCC activity as monitored by electrophysiological techniques. MCC may function in tandem with its outer membrane counterpart, a channel called PSC (for peptide-sensitive channel). If the outer and inner channels interact during protein translocation, then structural changes made to the outer membrane complex may also affect MCC activity. Therefore, similar antibody and proteolysis treatments of the outer membrane complex will be analyzed for effects on MCC activity. Biochemical reconstitution of MCC activity from genetically manipulated mitochondria will be used to identify protein components of the MCC. These components will then be analyzed by mass spectrometry to determine their amino acid sequences. Together the studies will provide fundamental new information on the mechanisms of protein import into mitochondria.
