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.

Abstract

The filamentous fungus Rhizopus nigricans responds to treatment with progesterone by inducing P450-associated redox carriers. Selection for azole resistance following expression of a cDNA library constructed with RNA from progesterone-treated R. nigricans in the yeast Saccharomyces cerevisiae led to the identification of CPR1-FL and CYB5-1 cDNAs, which code for functionally competent NADPH-cytochrome P450 reductase and cytochrome b5, respectively. 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 CYBS-1 and CPR2-CS mRNAs was not significantly affected. Analysis of the subcellular localization and function of the R. nigricans reductase in yeast indicated that the CPR1-FL cDNA and a derivative (CPR1-S) truncated at the first ATG codon gave rise to functionally equivalent products that were found in both cytosolic and microsomal fractions. In contrast, addition of an in-frame initiation codon at the 5' end of the CPR1-FL sequence resulted in localization of the activity mainly to the microsomes, and improved ketoconazole resistance but decreased NADPH-cytochrome c reductase activity in the host strain. These findings suggest that, of the three genes for P450-associated redox carriers investigated, only CPR1-FL is associated with the progesterone response and that its major transcript encodes a reductase that shows an unusual pattern of subcellular localization.