This Article Full Text Full Text (PDF) Supplemental material Other Versions of this Article: EC.00315-06v1 6/3/443    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pasrija, R. Articles by Prasad, R. Search for Related Content PubMed PubMed Citation Articles by Pasrija, R. Articles by Prasad, R.

 Previous Article  |  Next Article 

Eukaryotic Cell, March 2007, p. 443-453, Vol. 6, No. 3
1535-9778/07/$08.00+0     doi:10.1128/EC.00315-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Structure and Function Analysis of CaMdr1p, a Major Facilitator Superfamily Antifungal Efflux Transporter Protein of Candida albicans: Identification of Amino Acid Residues Critical for Drug/H⁺ Transport ^,

Membrane Biology Lab, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India

Received 3 October 2006/ Accepted 21 December 2006

We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent protein-tagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X₆)G(X₃)GP(X₂)GP(X₂)G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.

Published ahead of print on 5 January 2007.

Supplemental material for this article may be found at http://ec.asm.org/.

This article has been cited by other articles:

• Cowen, L. E., Steinbach, W. J. (2008). Stress, Drugs, and Evolution: the Role of Cellular Signaling in Fungal Drug Resistance. Eukaryot Cell 7: 747-764 [Full Text]  
• Pasrija, R., Panwar, S. L., Prasad, R. (2008). 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. Antimicrob. Agents Chemother. 52: 694-704 [Abstract] [Full Text]