AP1-mediated Multidrug Resistance in Saccharomyces cerevisiae Requires FLR1 Encoding a Transporter of the Major Facilitator Superfamily

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.

Thioredoxin Deficiency Causes the Constitutive Activation of Yap1, an AP-1-like Transcription Factor in Saccharomyces cerevisiae

“…Yap1 is constitutively localized in the nucleus in the trx1⌬/trx2⌬ mutant. GFP fluorescence was visualized in living yeast cells carrying pRS cp-GFP-YAP1 (25). Cells at exponential phase were harvested, resuspended in fresh SD medium, and treated with or without 0.2 mM H 2 O 2 .…”

“…The N terminus of Yap1 contains a bZip domain, which is conserved among the AP-1 family, including mammalian Jun, Fos, and S. cerevisiae Gcn4 (23). A cysteinerich domain (CRD) at the C terminus of Yap1, containing three Cys-Ser-Glu sequence motifs, plays an important role in the control of Yap1, especially for the intracellular localization of Yap1 (25,26). In the response to oxidative stress, the localization of Yap1 changes dramatically, while the increase in the DNA binding activity is modest and the levels of Yap1 do not increase (26 -28).…”

Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

“…Of the 16 genes that have previously been shown to be controlled by one or more of the five transcription factors, we counted six transporters, of which four were members of the ATP-binding cassette (ABC) transporter, PDR5 (Balzi et al, 1994), PDR10 (Wolfger et al, 1997), PDR15 (Wolfger et al, 2004) SNQ2 (Servos et al, 1993), and the remaining two were member of the major facilitator superfamilies TPO1 (Tomitori et al, 1999) and FLR1 (Alarco et al, 1997). The 11 remaining genes that were identified in this search do not encode multidrug resistance transporters but have other or still unknown functions.…”

YAP4 gene expression is induced in response to several forms of stress in Saccharomyces cerevisiae

“…Several studies have demonstrated that Yap1p, a b-Zip protein belonging to the yeast Yap family of transcriptional regulators (Rodrigues-Pousada et al, 2004), plays a central role in sensing and modulating gene expression in response to several forms of oxidative stress (Alarco et al, 1997;Delaunay et al, 2000;Gasch et al, 2000;Lee et al, 1999;Dumond et al, 2000;Nguyen et al, 2001;Wemmie et al, 1994;1366 T. Nevitt, J. Pereira and C. Rodrigues-Pousada Azevedo et al, 2003). Yap1p-responsive genes typically contain within their promoter region a Yap-response element (YRE-TTAC/GTAA) encoding for most cellular proteins with antioxidant function and several membrane-associated drug efflux pumps .…”