Import of proteins into mitochondria. Isolated yeast mitochondria and a solubilized matrix protease correctly process cytochrome c oxidase subunit V precursor at the NH2 terminus.

Abstract

The cytoplasmically made subunit V was isolated from enzymically active yeast cytochrome c oxidase and its NH2-terminal amino acid sequence was determined to be (formula; see text) In order to exclude that this NH2 terminus had been generated by proteolysis during the lengthy isolation of the subunit, subunit V was directly immunoprecipitated from yeast cells that had been pulse-labeled with [35S]methionine; radiochemical sequencing revealed methionine at position 12, in agreement with the sequence given above. When the precursor to subunit V was synthesized in vitro in the presence of either [35S]methionine, [3H]leucine, or [3H]histidine and then incubated either with isolated yeast mitochondria or the partially purified matrix protease (Böhni, P. C., Daum, G., and Schatz, G. (1983) J. Biol. Chem. 258, 4937-4943), it was converted to a polypeptide co-migrating with mature subunit V on dodecyl sulfate-polyacrylamide gels. Radiochemical sequence analysis of the processed in vitro product showed that it contained histidine, leucine, and methionine in positions 4, 6, and 12, respectively, exactly as the authentic mature protein. In contrast, the unprocessed precursor contained methionine only at position 9, but not at position 12; thus, the precursor has a NH2 terminus different from the mature polypeptide. Similarly, if the in vitro synthesized cytochrome b2 precursor is incubated with isolated mitochondria, it is converted to a polypeptide which co-migrates with mature cytochrome b2 and, like the latter, contains leucine and methionine in positions 4 and 6, respectively. These data show that isolated yeast mitochondria convert the precursors to polypeptides which have the NH2 terminus of the authentic mature polypeptides. In the case of cytochrome c oxidase subunit V, correct NH2-terminal processing was also demonstrated with the purified matrix protease.