This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shukla, S.
Right arrow Articles by Prasad, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shukla, S.
Right arrow Articles by Prasad, R.

 Previous Article  |  Next Article 

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.



This article has been cited by other articles:

  • Cannon, R. D., Lamping, E., Holmes, A. R., Niimi, K., Baret, P. V., Keniya, M. V., Tanabe, K., Niimi, M., Goffeau, A., Monk, B. C. (2009). Efflux-Mediated Antifungal Drug Resistance. Clin. Microbiol. Rev. 22: 291-321 [Abstract] [Full Text]  
  • Kolaczkowski, M., Kolaczkowska, A., Motohashi, N., Michalak, K. (2009). New High-Throughput Screening Assay To Reveal Similarities and Differences in Inhibitory Sensitivities of Multidrug ATP-Binding Cassette Transporters. Antimicrob. Agents Chemother. 53: 1516-1527 [Abstract] [Full Text]  
  • Tsao, S., Rahkhoodaee, F., Raymond, M. (2009). Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance. Antimicrob. Agents Chemother. 53: 1344-1352 [Abstract] [Full Text]  
  • Sauna, Z. E., Bohn, S. S., Rutledge, R., Dougherty, M. P., Cronin, S., May, L., Xia, D., Ambudkar, S. V., Golin, J. (2008). Mutations Define Cross-talk between the N-terminal Nucleotide-binding Domain and Transmembrane Helix-2 of the Yeast Multidrug Transporter Pdr5: POSSIBLE CONSERVATION OF A SIGNALING INTERFACE FOR COUPLING ATP HYDROLYSIS TO DRUG TRANSPORT. J. Biol. Chem. 283: 35010-35022 [Abstract] [Full Text]  
  • Manoharlal, R., Gaur, N. A., Panwar, S. L., Morschhauser, J., Prasad, R. (2008). Transcriptional Activation and Increased mRNA Stability Contribute to Overexpression of CDR1 in Azole-Resistant Candida albicans. Antimicrob. Agents Chemother. 52: 1481-1492 [Abstract] [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]  
  • Pasrija, R., Banerjee, D., Prasad, R. (2007). 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. Eukaryot Cell 6: 443-453 [Abstract] [Full Text]  
  • Saini, P., Gaur, N. A., Prasad, R. (2006). Chimeras of the ABC drug transporter Cdr1p reveal functional indispensability of transmembrane domains and nucleotide-binding domains, but transmembrane segment 12 is replaceable with the corresponding homologous region of the non-drug transporter Cdr3p.. Microbiology 152: 1559-1573 [Abstract] [Full Text]  
  • Prasad, T., Saini, P., Gaur, N. A., Vishwakarma, R. A., Khan, L. A., Haq, Q. M. R., Prasad, R. (2005). Functional Analysis of CaIPT1, a Sphingolipid Biosynthetic Gene Involved in Multidrug Resistance and Morphogenesis of Candida albicans. Antimicrob. Agents Chemother. 49: 3442-3452 [Abstract] [Full Text]  
  • Saini, P., Prasad, T., Gaur, N. A., Shukla, S., Jha, S., Komath, S. S., Khan, L. A., Haq, Q. Mohd. R., Prasad, R. (2005). Alanine scanning of transmembrane helix 11 of Cdr1p ABC antifungal efflux pump of Candida albicans: identification of amino acid residues critical for drug efflux. J Antimicrob Chemother 56: 77-86 [Abstract] [Full Text]  
  • Pasrija, R., Krishnamurthy, S., Prasad, T., Ernst, J. F., Prasad, R. (2005). Squalene epoxidase encoded by ERG1 affects morphogenesis and drug susceptibilities of Candida albicans. J Antimicrob Chemother 55: 905-913 [Abstract] [Full Text]  
  • Shukla, S., Ambudkar, S. V., Prasad, R. (2004). Substitution of threonine-1351 in the multidrug transporter Cdr1p of Candida albicans results in hypersusceptibility to antifungal agents and threonine-1351 is essential for synergic effects of calcineurin inhibitor FK520. J Antimicrob Chemother 54: 38-45 [Abstract] [Full Text]  
  • Mukhopadhyay, K., Prasad, T., Saini, P., Pucadyil, T. J., Chattopadhyay, A., Prasad, R. (2004). Membrane Sphingolipid-Ergosterol Interactions Are Important Determinants of Multidrug Resistance in Candida albicans. Antimicrob. Agents Chemother. 48: 1778-1787 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»