Localization of a synthetic presequence that blocks protein import into mitochondria

In the accompanying paper (Glaser, S. M., and Cumsky, M. G. (1990) J. Biol. Chem. 265, 8808-8816) we demonstrated that pL4-(1-22), a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence, blocked protein import into mitochondria. Import inhibition was reversible and occurred at a step subsequent to the initial recognition and binding of precursor proteins to the mitochondrial surface. In the present work we have studied the nature of the association between the peptide and mitochondria, as well as determined its intramitochondrial location. We found that pL4-(1-22) was imported into mitochondria in a manner that was dependent upon the delta psi and that the majority of the mitochondrially associated peptide was in the membrane fraction. Density gradient analysis of total membranes indicated that pL4-(1-22) cofractionated with the inner membrane, although the possibility that it was present in both membranes could not be ruled out. It appeared to be inserted within the bilayer since it could not be extracted with salts, chaotropic agents, or high pH. We observed a steady decrease in the amount of pL4-(1-22) found within peptide-treated mitochondria over time. Coincident with this decrease was an increase in the ability of those mitochondria to import and process precursor proteins, suggesting that the peptide was ultimately turned over. The results presented here correlate well with those of the accompanying paper. Together they suggest that pL4-(1-22) blocks import at the level of the mitochondrial membranes, although the exact nature of the import block is not yet clear.