Abstract
In order to study the role of protein unfolding during post-translational protein import into mitochondria, we destabilized the structure of a mitochondrial precursor protein by site-directed mutagenesis. The precursor consisted of the first 16 residues of the yeast cytochrome oxidase subunit IV precursor fused to mouse dihydrofolate reductase. Labilization of the folded precursor structure was monitored by increased susceptibility to protease and diminished ability of methotrexate to block import of the precursor into isolated yeast mitochondria. On comparing the original precursor with two mutant forms that were destabilized to different degrees, increased labilization correlated with an increased rate and efficiency of import into mitochondria. This supports the view that the precursor must unfold in order to enter the mitochondria.