Import of proteins into mitochondria. Energy-dependent uptake of precursors by isolated mitochondria.

Abstract

The post-translational import of cytoplasmically synthesized precursors of proteins of the mitochondrial matrix and inner membrane has been studied in vitro with isolated yeast mitochondria and reticulocyte lysates programmed with yeast mRNA. Import of precursors into isolated mitochondria is not a nonspecific side reaction: up to 70% of the in vitro-synthesized precursor of the &subunit of FIATPase can be processed to the mature form and rendered protease-resistant during incubation with mitochondria. Furthermore, precursors which were processed to their mature form prior to incubation with mitochondria do not become protease-resistant under similar conditions. Finally, a brief trypsin treatment of mitochondria prior to incubation with labeled F1 psubunit precursor abolished uptake of this precursor into the mitochondria. In vitro import occurs into the correct intramitochondrial location: this was determined by the use of chelators such as GTP and bathophenanthroline disulfonate which inhibit the cleavage of precursors to their mature form by a hypotonic extract from yeast mitochondria or by lysed mitochondria. The processing of these precursors by intact mitochondria, however, is not affected by such inhibitors. Since these inhibitors can cross the outer, but not the inner, mitochondrial membrane, at least part of the precursor molecule is translocated across an intact inner membrane before cleavage to the mature form occurs. Subfractionation of mitochondria after in vitro import provides further evidence for the proper localization of proteins imported into the mitochondrial matrix, intermembrane space, and membrane fraction. Import of precursors into isolated yeast mitochondria thus exhibits the topological specificity of the process as it occurs in living cells. The in vitro import of precursor polypeptides requires energy: in respiring mitochondria, import is blocked either by protonophores or by valinomycin plus potassium, but not by oligomycin; in cyanide-inhibited mitochondria supplemented with external ATP, import is blocked by carboxyatractyloside or by oligomycin. This shows that an electrochemical gradient across the inner mitochondrial membrane, and not ATP itself, is required for protein import.