Synthesis and Some Aspects of Topogenesis of Bovine Cytochrome P450scc in Yeast

A. S. Savelev,¹ L. A. Novikova,¹ V. L. Drutsa,¹ L. V. Isaeva,¹ A. A. Chernogolov,² S. A. Usanov,² and V. N. Luzikov^1,3

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-28-76; E-mail: luzikov@beloz.genebee.msu.su

²Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Zhodinskaya ul. 5/2, Minsk, 220141 Belarus; fax: (375-172) 63-72-74; E-mail: usanov@eco2.iasnet.com

³To whom correspondence should be addressed.

Submitted March 3, 1997; revision submitted April 4, 1997.

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A plasmid for effective expression of recombinant DNA encoding a hybrid protein composed of the N-terminal targeting presequence of subunit IV of yeast cytochrome c oxidase preceding the mature polypeptide chain of bovine cytochrome P450scc (pCoxIV-CYP11A1) in yeast has been constructed. It has been shown that this protein, when synthesized in yeast cells, is imported into mitochondria and undergoes proteolytic processing, thus yielding a product of molecular mass corresponding to that of mature cytochrome P450scc. However, only insignificant portion of the imported protein proves to be inserted into the inner membrane of heterologous mitochondria. The membrane-bound cytochrome P450scc exhibits cholesterol hydroxylase activity towards 22R-hydroxycholesterol in the presence of exogenous adrenodoxin and adrenodoxin reductase. This fact indicates that the foreign protein is correctly folded and oriented in the membrane. Thus, insertion into the inner membrane is a limiting step of the pCoxIV-CYP11A1 topogenesis in yeast cells, whereas its import into mitochondria and proteolytic processing proceed without significant impediments.

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KEY WORDS: cytochrome P450scc, yeast, synthesis, mitochondria, import, processing, location, cholesterol hydroxylase activity.

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