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Eukaryotic Cell, December 2003, p. 1361-1375, Vol. 2, No. 6
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.6.1361-1375.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Functional Characterization of Candida albicans ABC Transporter Cdr1p

Suneet Shukla,1 Preeti Saini,1 Smriti,1,{dagger} Sudhakar Jha,1 Suresh V. Ambudkar,2 and Rajendra Prasad1*

Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India,1 Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208922

Received 21 July 2003/ Accepted 7 October 2003

In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and {Delta}F774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion ({Delta}F774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized {Delta}F774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).

* Corresponding author. Mailing address: Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India. Phone: 91-11-26704509. Fax: 91-11-26717081. E-mail: rp47{at}hotmail.com.

{dagger} Present address: Department of Biochemistry and Molecular Biology, School of Medicine, Indiana University, Bloomington, IN 47405.

Eukaryotic Cell, December 2003, p. 1361-1375, Vol. 2, No. 6
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.6.1361-1375.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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Copyright © 2003 by the American Society for Microbiology.