Shukla, Suneet ; Saini, Preeti ; Smriti, ; Jha, Sudhakar ; Ambudkar, Suresh V. ; Prasad, Rajendra (2003) Functional characterization of Candida albicans ABC transporter Cdr1p Eukaryotic Cell, 2 (6). pp. 1361-1375. ISSN 1535-9778
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Official URL: http://ec.asm.org/cgi/content/abstract/2/6/1361
Related URL: http://dx.doi.org/10.1128/EC.2.6.1361-1375.2003
Abstract
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 ΔF774 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 (ΔF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ΔF774 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).
Item Type: | Article |
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Source: | Copyright of this article belongs to American Society for Microbiology. |
ID Code: | 39357 |
Deposited On: | 12 May 2011 05:41 |
Last Modified: | 17 May 2016 21:50 |
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