RESUMO
We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Ferredoxina-NADP Redutase/química , Mitocôndrias/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Adrenodoxina/química , Adrenodoxina/genética , Adrenodoxina/metabolismo , Animais , Domínio Catalítico , Bovinos , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/metabolismo , Humanos , Immunoblotting , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Pregnenolona/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/química , Esteroide Hidroxilases/química , Esteroide Hidroxilases/metabolismoRESUMO
When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pim1p protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pim1p, respectively. These data imply that chaperones and Pim1p protease prefer their natural targets in mitochondria to imported foreign proteins.
