AMINOTRANSFERASE VARIANTS AS PROBES FOR THE ROLE OF THE N-TERMINAL REGION OF A MATURE PROTEIN IN MITOCHONDRIAL PRECURSOR IMPORT AND PROCESSING

Of the two homologous isozymes of aspartate aminotransferase that are also nearly identical in their folded structures, only the mitochondri al form (mAAT) is synthesized as a precursor (pmAAT). After its in vit ro synthesis in rabbit reticulocyte lysate, it can also be efficiently imported into isolated rat liver mitochondria, where it is processed to its native form by removal of the N-terminal presequence. The homol ogous cytosolic isoenzyme (cAAT) is not imported into mitochondria, ev en after fusion of the mitochondrial presequence from pmAAT to its N-t erminal end. Substitution of the 30-residue N-terminal peptide of the mature portion of pmAAT with the corresponding sequence from the homol ogous, import-incompetent cytosolic isozyme (pc-mAAT) does not prevent import but reduces substantially its processing in the matrix. A dete ctable amount of the pcmAAT chimera is found associated with the inner mitochondrial membrane. Single and double substitution mutants of Trp -5 and Trp-6 at the N-terminal end of the mature protein are imported into mitochondria with efficiency similar to that of wild type. Howeve r, replacement of Trp-5 with proline, or of both tryptophans with eith er alanine (W5A/W6A mutant) or valine and aline (W5V/W6A mutant), allo ws import but interferes with the correct processing of the imported p rotein despite the presence of an intact cleavage site for the process ing peptidase. Similar cleavage results were obtained using newly synt hesized proteins and mitochondrial matrix extracts. These results indi cate that translocation and processing for a precursor are independent events and that sequences C-terminal to the cleavage site are indeed important for the correct maturation of pmAAT in the matrix, probably because of their contribution to the conformation and flexibility of t he peptide region surrounding the cleavage site required for efficient processing. The same region from the mature component of the passenge r protein to complete its translocation into the matrix.