Organella-targeted expression of rat liver cytochrome P450c27 in yeast. Genetically engineered alteration of mitochondrial P450 into a microsomal form creates a novel functional electron transport chain.
A modified rat cytochrome P450c27, whose mitochondrial targeting signal had been replaced by a possible microsomal targeting signal of bovine cytochrome P450c17, was expressed in yeast. The modified P450c27 hemoprotein was correctly localized on yeast microsomes and exhibited the P450c27-dependent monooxygenase activity by addition of bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR). Considering the previous observation that P450c27 with its own mitochondrial targeting signal was imported into yeast mitochondria (Akiyoshi-Shibata, M., Usui, E., Sakaki, T., Yabusaki, Y., Noshiro, M., Okuda, K., and Ohkawa, H. (1991) FEBS Lett. 280, 367-370), it is now suggested that the destination of P450c27 to either mitochondria or microsomes in yeast depends solely on the amino-terminal targeting signal. In addition, the modified P450c27 was simultaneously expressed in yeast with mature forms of bovine ADX and ADR. The recombinant yeast produced the P450 on the microsomes and mature forms of ADX and ADR in the cytoplasm, and showed the monooxygenase activity. Accordingly, a novel type of functional electron transport chain has been established between the cytoplasm and the microsomes in yeast.
Sakaki
T
T
Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Hyogo, Japan.
Akiyoshi-Shibata
M
M
Yabusaki
Y
Y
Ohkawa
H
H
D000818:Animals; D015153:Blotting, Western; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D017886:Durapatite; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D006882:Hydroxyapatites; D007106:Immune Sera; D007700:Kinetics; D008854:Microscopy, Electron; D008861:Microsomes / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D015388:Organelles / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D006882:Hydroxyapatites; D007106:Immune Sera; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D006899:Mixed Function Oxygenases