Use of the addressing sequence of yeast D-lactate dehydrogenase for insertion of CYP11A1p into the inner membrane of yeast mitochondria

Mammalian cytochrome P450scc (CYP11A1p) is a pseudointegral protein of the inner membrane of mitochondria with the active center exposed in the matrix. Upon import of the CYP11A1p precursor into yeast mitochondria, only a minor part was incorporated into the inner mitochondrial membrane and acquired catalytic activity (Kovaleva, I. E., Novikova, L. A., Nazarov, P. A., Grivennikov, S. I., and Luzikov, V. N. (2003) Eur. J. Biochem., 270, 222-229). The present work is an attempt to increase the efficiency of this process by substitution of the inherent N-terminal presequence of CYP11A1p by the addressing signal of D-lactate dehydrogenase (D-LD) of the yeast Saccharomyces cerevisiae. D-LD is known to be inserted into the inner membrane of mitochondria through its transmembrane domain located close to the N-terminus of the polypeptide chain in such a way that the protein globule is exposed in the intermembrane space. The hybrid protein D-LD(1-72)-mCYP11A1p synthesized in yeast cells was imported into yeast mitochondria, underwent processing, and was inserted into the inner membrane on the side of the intermembrane space. In the presence of adrenodoxin and adrenodoxin reductase, the hybrid protein exhibited cholesterol side-chain cleavage activity. Thus, CYP11A1p insertion into the inner membrane of mitochondria mediated by the D-LD topogenic signal resulted in the catalytically active mCYP11A1p domain in the hybrid protein.