USE OF PROTEASE SENSITIVITY TO PROBE THE CONFORMATIONS OF NEWLY SYNTHESIZED MUTANT FORMS OF MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE

Sensitivity to digestion with pronase has been used to show that the p recursor form of mitochondrial aspartate aminotransferase, the form la cking the N-terminal presequence, that with a deletion of the first 9 residues and mutants of the mature enzyme in which residue Cys-166 is mutated to alanine or serine, all retain unfolded conformations after synthesis in a reticulocyte lysate. In the presence of lysed mitochond ria the various forms of mitochondrial aspartate aminotransferase reta ined their susceptibilities to pronase in a way that mirrored the effi ciencies with which they are imported into intact mitochondria. The re sults are interpreted as showing that the presequence of mitochondrial aspartate aminotransferase is not uniquely required for interaction w ith cytosolic factors required to maintain the newly synthesised prote in in a form competent for interacting with, and being imported into, mitochondria. (C) 1995 Academic Press, Inc.