Effects of various N‐terminal addressing signals on sorting and folding of mammalian CYP11A1 in yeast mitochondria

Topogenesis of cytochrome P450scc, a resident protein of the inner membrane of adrenocortical mitochondria, is still obscure. In particular, little is known about the cause of its tissue specificity. In an attempt to clarify this point, we examined the process in Saccharomyces cerevisiae cells synthesizing cytochrome P450scc as its native precursor (pCYP11A1) or versions in which its N‐terminal addressing presequence had been replaced with those of yeast mitochondrial proteins: CoxIV(1–25) and Su9(1–112). We found the pCYP11A1 and CoxIV(1–25)‐mCYP11A1 versions to be effectively imported into yeast mitochondria and subjected to proteolytic processing. However, only minor portions of the imported proteins were incorporated into mitochondrial membranes, whereas their bulk accumulated as aggregates insoluble in 1% Triton X‐100. Along with previously published data, this suggests that a distinguishing feature of the import of the CYP11A1 precursors into yeast mitochondria is their easy translocation into the matrix where the foreign proteins mainly undergo proteolysis or aggregation. The fraction of CYP11A1 that happens to be inserted into the inner mitochondrial membrane is effectively converted into the catalytically active holoenzyme. Experiments with the Su9(1–112)‐mCYP11A1 construct bearing a re‐export signal revealed that, after translocation of the fused protein into the matrix and its processing, the Su9(67–112) segment ensures association of the mCYP11A1 body with the inner membrane, but proper folding of the latter does not take place. Thus it can be said that the most specific stage of CYP11A1 topogenesis in adrenocortical mitochondria is its confinement and folding in the inner mitochondrial membrane. In yeast mitochondria, only an insignificant portion of the imported CYP11A1 follows this mechanism.