Abstract
We have isolated a Candida albicans gene that confers resistance to the azole derivative fluconazole (FCZ) when overexpressed in Saccharomyces cerevisiae. This gene encodes a protein highly homologous to S. cerevisiae yAP-1, a bZip transcription factor known to mediate cellular resistance to toxicants such as cycloheximide (CYH), 4-nitroquinoline N-oxide (4-NQO), cadmium, and hydrogen peroxide. The gene was named CAP1, for C. albicans AP-1. Cap1 and yAP-1 are functional homologues, since CAP1 expression in a yap1 mutant strain partially restores the ability of the cells to grow on toxic concentrations of cadmium or hydrogen peroxide. We have found that the expression of YBR008c, an open reading frame identified in the yeast genome sequencing project and predicted to code for a multidrug transporter of the major facilitator superfamily, is dramatically induced in S. cerevisiae cells overexpressing CAP1. Overexpression of either CAP1 or YAP1 in a wild-type strain results in resistance to FCZ, CYH, and 4-NQO, whereas such resistance is completely abrogated (FCZ and CYH) or strongly reduced (4-NQO) in a ybr008c deletion mutant, demonstrating that YBR008c is involved in YAP1- and CAP1-mediated multidrug resistance. YBR008c has been renamed FLR1, for fluconazole resistance 1. The expression of an FLR1-lacZ reporter construct is strongly induced by the overexpression of either CAP1 or YAP1, indicating that the FLR1 gene is transcriptionally regulated by the Cap1 and yAP-1 proteins. Taken collectively, our results demonstrate that FLR1 represents a new YAP1-controlled multidrug resistance molecular determinant in S. cerevisiae. A similar detoxification pathway is also likely to operate in C. albicans.
Highlights
Cells have evolved elaborate molecular mechanisms to protect themselves from injuries caused by environmental exposure to toxic compounds of different structures and functions
We found that the three genes (YAP1, CAP1, and CAP1-TR) were able to confer FCZ resistance when expressed in the wild-type YBR008c strain, demonstrating that the ability of CAP1 to mediate FCZ resistance in S. cerevisiae is not restricted to the C. albicans gene and extends to its S. cerevisiae homologue
Investigation of C. albicans molecular determinants of FCZ resistance has allowed us to isolate a gene coding for a new member of the yeast AP-1 family that we have named CAP1
Summary
Yeast Strains and Media—S. cerevisiae diploid strain W303 (a/␣ ade2/ade his3/his leu2/leu trp1/trp ura3/ura can1/can1) was a gift from M. Drug Resistance Assays—For microtiter plate assays, cells grown for 48 h on selective SD Ϫura were resuspended in a saline solution (0.85%) to an A600 of 0.1. For the cadmium resistance spot assay, transformants were grown overnight in SD Ϫura medium and approximately 104 cells were spotted onto a YPD plate containing 0 or 10 M cadmium. -Galactosidase Assays—An FLR1(YBR008c)-lacZ fusion plasmid was constructed using a PCR fragment overlapping the promoter region, the translation initiation codon, as well as a short portion of the coding region of the FLR1 gene (positions Ϫ828 to ϩ25) This PCR fragment was generated using oligonucleotides 5Ј-CGGGATCCGGTAGAAGAGTTACGGAA and 5Ј-CCAAGCTTTGTCTGTACGTTGAAGTGTA, which introduce, respectively, a 5Ј BamHI and a 3Ј HindIII site for directional cloning into YEp368 cleaved with BamHI and HindIII [62], generating plasmid YEp368/FLR1. Protein concentrations were determined by the method of Bradford [64], using bovine serum albumin as standard
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