Characterization of Cdr1p, A Major Multidrug Efflux Protein of Candida albicans:  Purified Protein Is Amenable to Intrinsic Fluorescence Analysis

Abstract

Candida drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, confers multidrug resistance in immunocompromised and debilitated patients. A member of the ATP-binding cassette (ABC) superfamily of membrane transporters, Cdr1p contains two nucleotide binding/utilization sites (NBDs) and two transmembrane domains (TMDs). We had earlier characterized Cdr1p by its overexpression as a GFP-tagged fusion protein that elicits oligomycin-sensitive ATPase activity and is linked to drug extrusion. However, it is essential to have highly purified Cdr1p to understand the detailed molecular basis of structure and functions of this protein. In this study, we have developed a two-step purification protocol using stably overexpressed His-tagged Cdr1p in Saccharomyces cerevisiae. Purified Cdr1p exhibited divalent cation-dependent ATPase activity [approximately 1.2 micromol (mg of protein)(-)(1) min(-)(1)] with an apparent K(M) in the range of 1.8 to 2.1 mM and V(max) between 1.0 and 1.4 micromol (mg of protein)(-)(1) min(-)(1). Unlike its close homologue human P-gp/MDR1, purified Cdr1p only moderately displayed drug stimulated ATPase activity. By exploiting intrinsic fluorescence intensity of purified Cdr1p, which contains 24 tryptophan residues, we could monitor defined conformational changes upon substrate drug and ATP binding. It is observed that ATP binding to Cdr1p (K(d) = approximately 1.7 mM) is not a prerequisite for drug binding, and both the mechanisms of drug as well as ATP binding, which induce specific conformational changes, occur independent of each other. Our study for the first time provides a catalytically active purified ABC transporter from a fungal pathogen, which is amenable to fluorescence measurements and thus would be useful in understanding the molecular basis of antifungal transport.

Curcumin Modulates Efflux Mediated by Yeast ABC Multidrug Transporters and Is Synergistic with Antifungals

“…We had shown previously that IAAP (a photoaffinity analogue of the human Pglycoprotein substrate prazosin) and azidopine (a dihydropyridine photoaffinity analogue of its modulator, verapamil) specifically bind to CaCdr1p (27 (15,26,27), CUR at 100 M had no effect on ATPase activity in the presence of varying concentrations of ATP (Fig. 4C).…”

Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

“…We also found that (40), and (iii) Cdr1p-and Cdr2p-mediated transport across the plasma membranes of whole S. cerevisiae cells was known to be ATP dependent (11,21,49 (20). Two possible explanations for these differing results are that (i) other vanadate-sensitive transporters may also pump […”

Multidrug Transporters CaCdr1p and CaMdr1p of Candida albicans Display Different Lipid Specificities: both Ergosterol and Sphingolipids Are Essential for Targeting of CaCdr1p to Membrane Rafts

“…In this study, we exploited the well-established and extensively used expression system of S. cerevisiae for the overexpression of CaCdr1p and CaMdr1p (10,25,35,42,43). The strategy of GFP tagging and cloning of CaCdr1p and CaMdr1p in the plasmid pSK-PDR5PPUS was described previously (35,43).…”