PUROMYCIN INHIBITS PROTEIN IMPORT INTO MITOCHONDRIA BY INTERFERING WITH AN INTRAMITOCHONDRIAL ATP-DEPENDENT REACTION

We have performed experiments which demonstrate that puromycin inhibit s the import of proteins into mitochondria in in vitro reactions conta ining mitochondria isolated from the yeast Saccharomyces cerevisiae an d precursor proteins synthesized in a nuclease-treated rabbit reticulo cyte lysate. Puromycin inhibited the import of several precursor prote ins including; a fusion protein consisting of the first 22 N-terminal residues of yeast cytochrome oxidase subunit IV fused to mouse dihydro folate reductase, both a destabilized and truncated form of this same fusion protein, the beta-subunit of the yeast mitochondrial F1-ATPase and yeast alcohol dehydrogenase III. The insertion of the yeast outer mitochondrial protein porin was not inhibited by puromycin. Puromycin- induced import inhibition could be overcome by adding additional ATP t o the import reactions. However, if access of ATP to the mitochondrial matrix was prevented by blocking the adenine nucleotide translocase w ith carboxyatractyloside, ATP addition was unable to overcome the inhi bitory effect of puromycin on protein import. Collectively, these resu lts demonstrate that puromycin inhibits protein import into mitochondr ia by interfering with an ATP-dependent step in the import process and that the ATP-dependent component in the reaction is located inside th e inner mitochondrial membrane. In addition to supporting the view tha t ATP is required in the matrix for efficient protein import, these re sults may provide a useful tool for identifying the ATP-binding compon ents of the import apparatus.