Functional cloning, based on azole resistance in Saccharomyces cerevisiae,and characterization of Rhizopus nigricans redox carriers that are differentially involved in the P450-dependent response to progesterone stress

The filamentous fungus Rhizopus nigricans responds to treatment with proges terone by inducing P450-associated redox carriers. Selection for azole resi stance following expression of a cDNA library constructed with RNA from pro gesterone-treated R. nigricans in the yeast Saccharomyces cerevisiae led to the identification of CPR1-FL and CYB5-1 cDNAs, which code for functionall y competent NADPH-cytochrome P450 reductase and cytochrome b(5), respective ly. The central region (CPR2-CS) of an additional reductase gene sharing 66 % identity with CPR1-FL was cloned from progesterone-induced mRNA by RT-PCR , using primers based on consensus sequences. Northern analysis of the 2.1- kb transcripts revealed that, of the two cloned reductase genes, only CPR1- FL mRNA was strongly induced by progesterone; transcription of CYB5-1 and C PR2-CS mRNAs was not significantly affected. Analysis of the subcellular lo calization and function of the R. nigricans reductase in yeast indicated th at the CPR1-FL cDNA and a derivative (CPR1-S) truncated at the first ATG co don gave rise to functionally equivalent products that were found in both c ytosolic and microsomal fractions. In contrast, addition of an in-frame ini tiation codon at the 5 ' end of the CPR1-FL sequence resulted in localizati on of the activity mainly to the microsomes, and improved ketoconazole resi stance but decreased NADPH-cytochrome c reductase activity in the host stra in. These findings suggest that, of the three genes for P450-associated red ox carriers investigated, only CPR1-FL is associated with the progesterone response and that its major transcript encodes a reductase that shows an un usual pattern of subcellular localization.