Characterization of Three Classes of Membrane Proteins Involved in Fungal Azole Resistance by Functional Hyperexpression inSaccharomyces cerevisiae

Abstract

The study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomal PDR5 locus of a modified host Saccharomyces cerevisiae strain, AD⌬. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, His, FLAG/His, Cys, or His/Cys tags. Most GFP-tagged proteins tested were correctly trafficked within the cell, and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the apparent K m value and the V max value of ATPase activities were not significantly affected by the addition of His tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.The resolution and exploitation of protein structure and function are among the greatest biological challenges in the postgenomic era. These challenges, and their potential dividends, are greatest for membrane proteins, which are notoriously difficult to functionally express and purify in the quantities and forms needed for drug discovery or for high-resolution X-ray crystallography (1, 16). About a quarter of the cellular proteome consists of membrane proteins (5), which often play vital physiological roles: from environmental sensing to energy transduction, from nutrient uptake to drug efflux, and from cellular proliferation to programmed cell death. Membrane proteins are involved in many prominent diseases, including cystic fibrosis (48), type 2 diabetes (49), heart disease (52), and the drug resistance of numerous cancers (57). Hence, they are the targets for many therapies and constitute up to 70% of the drug targets used in medicine today. Membrane proteins also play key roles in drug modification, detoxification, and resistance in a wide variety of prokaryotic and eukaryotic systems (7). A fundamental understanding of cell biology, cell physiology, and cell-drug interactions therefore requires a detailed analysis of membrane protein function. Fu...

The roles of CDR1, CDR2, and MDR1 in kaempferol-induced suppression with fluconazole-resistant Candida albicans

“…The upregulations of multidrug efflux pump controlled by Cdr1p, Cdr2p belonging to ATP-binding cassette superfamily (APC transporter) and Mdr1p, a member of major facilitator superfamily (MFS) were implicated in most fluconazole-resistant C. albicans strains (Niimi et al 2004;Holmes et al 2012;Prasad and Rawal 2014) as FLC was a substrate for CDR1, CDR2, and MDR1 (Kohli et al 2001;Nakamura et al 2001). Rhodamine 6G was the most common used fluorescent dye of ABC pump substrate requiring ATP as energy to observe transporter-mediated FLC resistance (Lamping et al 2007;Peralta et al 2012). Our experiments showed that FLC alone failed to lead to rhodamine 6G accumulation in the intracellular space in C. albicans z2003 due to active efflux pump (Figure 3).…”

Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance

“…Because of the advanced genetics of S. cerevisiae, most studies of Cdr1p and Cdr2p have been carried out with heterologous expression systems in S. cerevisiae, where Cdr1p and Cdr2p were expressed under the control of a strong promoter, leading to very high levels of azole resistance (ϳ100-fold) (12,31). It was shown that the two transporters localize at the plasma membrane (35,37), bind rhodamine 6G (R6G) (12), export their substrates in an energy-dependent manner, and possess ATPase and phospholipid translocase activities (15,39). Expression systems in S. cerevisiae have also proved useful for structure-function studies of the transmembrane and ATPbinding domains of Cdr1p (14,26,31).…”

“…First, the function of ABC transporters is influenced by their lipid environment (29,40) and, since S. cerevisiae and C. albicans have different plasma membrane lipid compositions (17,42), the function of Cdr1p and Cdr2p could be altered in S. cerevisiae. Also, the S. cerevisiae strains used for these studies carry deletions in (up to seven) genes which encode endogenous ABC transporters (12,15). Since these ABC transporters can function as sterol transporters or phospholipid flippases (8,27), the deletion of these genes may affect the plasma membrane composition of the recipient yeast and thus Cdr1p and Cdr2p function.…”

Resistance reversal induced by a combination of fluconazole and tacrolimus (FK506) in Candida glabrata

“…Moreover, further functional assays confirmed that FK506 reduced the efflux of FLC. Additionally, another study indicated that FK506 can enhance the antifungal effect of FLC against a strain overexpressing the CDR1 gene in C. glabrata (Lamping et al, 2007). These results demonstrate that a combination of FLC and FK506 may act synergistically against C. glabrata at both the gene and protein levels.…”