1887

Abstract

The molecular basis of the broad substrate recognition and the transport of substrates by Cdr1p, a major drug efflux protein of , is not well understood. To investigate the role of transmembrane domains and nucleotide-binding domains (NBDs) of Cdr1p in drug transport, two sets of protein chimeras were constructed: one set between homologous regions of Cdr1p and the non-drug transporter Cdr3p, and another set consisting of Cdr1p variants comprising either two N- or two C-terminal NBDs of Cdr1p. The replacement of either the N- or the C-terminal half of Cdr1p by the homologous segments of Cdr3p resulted in non-functional recombinant strains expressing chimeric proteins. The results suggest that the chimeric protein could not reach the plasma membrane, probably because of misfolding and subsequent cellular trafficking problems, or the rapid degradation of the chimeras. As an exception, the replacement of transmembrane segment 12 (TMS12) of Cdr1p by the corresponding region of Cdr3p resulted in a functional chimera which displayed unaltered affinity for all the tested substrates. The variant protein comprising either two N-terminal or two C-terminal NBDs of Cdr1p also resulted in non-functional recombinant strains. However, the N-terminal NBD variant, which also showed poor cell surface localization, could be rescued to cell surface, if cells were grown in the presence of drug substrates. The rescued chimera remained non-functional, as was evident from impaired ATPase and efflux activities. Taken together, the results suggest that the two NBDs of Cdr1p are asymmetric and non-exchangeable and that the drug efflux by Cdr1p involves complex interactions between the two halves of the protein.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28471-0
2006-05-01
2019-11-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/5/1559.html?itemId=/content/journal/micro/10.1099/mic.0.28471-0&mimeType=html&fmt=ahah

References

  1. Aleksandrov, L., Aleksandrov, A. A., Chang, X. & Riordan, J. R. ( 2002; ). The first nucleotide-binding domain of CFTR is a site of stable nucleotide intercation whereas the second is a site of rapid turnover. J Biol Chem 277, 15419–15425.[CrossRef]
    [Google Scholar]
  2. Balan, I., Alarco, A. M. & Raymond, M. ( 1997; ). The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter. J Bacteriol 179, 7210–7218.
    [Google Scholar]
  3. Beaudet, L. & Gros, P. ( 1995; ). Functional dissection of P-glycoprotein nucleotide binding domains in chimeric and mutant proteins. J Biol Chem 270, 17159–17170.[CrossRef]
    [Google Scholar]
  4. Braun, B. R., Hoog, M. V. H., d'Enfert, C. & 40 other authors ( 2005; ). A human curated annotation of Candida albicans genome. PLoS Genet 1, 36–57.[CrossRef]
    [Google Scholar]
  5. Dogra, S., Krishnamurthy, S., Gupta, V., Dixit, B. L., Gupta, C. M., Sanglard, D. & Prasad, R. ( 1999; ). Asymmetric distribution of phosphatidylethanolamine in C. albicans: possible mediation by CDR1, A multidrug transporter belonging to ATP binding cassette (ABC) superfamily. Yeast 15, 111–121.[CrossRef]
    [Google Scholar]
  6. Franz, R., Michel, S. & Morschhauser, J. ( 1998; ). A fourth gene from the Candida albicans CDR family of ABC transporters. Gene 220, 91–98.[CrossRef]
    [Google Scholar]
  7. Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P. C. & Deeley, R. G. ( 2000; ). Comparison of the functional characteristics of the nucleotide binding domains of the multidrug resistance protein 1. J Biol Chem 275, 13098–13108.[CrossRef]
    [Google Scholar]
  8. Gaur, M., Devapriya, C. & Prasad, R. ( 2005; ). The complete inventory of ABC proteins in human pathogenic yeast, Candida albicans. J Mol Microbiol Biotechnol 9, 3–15.[CrossRef]
    [Google Scholar]
  9. Henriksen, U., Gether, U. & Litman, T. ( 2005; ). Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2. J Cell Sci 118, 1417–1426.[CrossRef]
    [Google Scholar]
  10. Hrycyna, C. A., Ramachandra, M., Germann, U. A., Cheng, P., Wu, Pastan, I. & Gottesman, M. M. ( 1999; ). Both ATP sites of human P-glycoprotein are essential but not symmetric. Biochemistry 38, 13887–13899.[CrossRef]
    [Google Scholar]
  11. Jha, S., Karnani, N., Dhar, S. K., Mukhopadhyay, K., Shukla, S., Saini, P., Mukhopadhyay, G. & Prasad, R. ( 2003a; ). Purification and charaterization of N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis. Biochemistry 42, 10822–10832.[CrossRef]
    [Google Scholar]
  12. Jha, S., Karnani, N., Lynn, A. M. & Prasad, R. ( 2003b; ). Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump. Biochem Biophys Res Commun 310, 869–875.[CrossRef]
    [Google Scholar]
  13. Jha, S., Dabas, N., Karnani, N., Saini, P. & Prasad, R. ( 2004; ). ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry. FEMS Yeast Res 5, 63–72.[CrossRef]
    [Google Scholar]
  14. Kohli, A., Smriti, Mukhopadhyay, K., Rattan, A. & Prasad, R. ( 2002; ). In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry. Antimicrob Agents Chemother 46, 1046–1052.[CrossRef]
    [Google Scholar]
  15. Krishnamurthy, S., Gupta, V., Snehlata, P. & Prasad, R. ( 1998a; ). Characterisation of human steroid hormone transport mediated by Cdr1p, multidrug transporter of Candida albicans, belonging to the ATP binding cassette super family. FEMS Microbiol Lett 158, 69–74.[CrossRef]
    [Google Scholar]
  16. Krishnamurthy, S., Chatterjee, U., Gupta, V., Prasad, R., Das, P., Snehlata, P., Hasnain, S. E. & Prasad, R. ( 1998b; ). Deletion of transmembrane domain 12 of CDR1, a multidrug transporter from Candida albicans, leads to altered drug specificity: expression of a yeast multidrug transporter in Baculovirus expression system. Yeast 14, 535–550.[CrossRef]
    [Google Scholar]
  17. Loo, T. W. & Clarke, D. M. ( 1997; ). Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators. J Biol Chem 272, 709–712.[CrossRef]
    [Google Scholar]
  18. Morello, J.-P., Petaja-Repo, U. E., Bichet, D. G. & Bouvier, M. ( 2000; ). Pharmacological chaperones: a new twist on receptor folding. Trends Pharmacol Sci 21, 466–468.[CrossRef]
    [Google Scholar]
  19. Mukhopadhyay, K., Kohli, A. K. & Prasad, R. ( 2002; ). Drug susceptibilities of yeast cells are affected by membrane lipid composition. Antimicrob Agents Chemother 46, 3695–3705.[CrossRef]
    [Google Scholar]
  20. Nakamura, K., Niimi, M., Niimi, K., Holmes, A. R., Yates, J. E., Decottignies, A., Monk, B. C., Goffeau, A. & Cannon, R. D. ( 2002; ). Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters. Antimicrob Agents Chemother 45, 3366–3374.
    [Google Scholar]
  21. Ng, W. F., Sarangi, F., Zastawny, R. L., Veinot-Drebot, L. & Ling, V. ( 1989; ). Identification of members of the P-glycoprotein multigene family. Mol Cell Biol 9, 1224–1232.
    [Google Scholar]
  22. Nikaido, K. & Ames, G. F. L. ( 1999; ). One intact ATP-binding subunit is sufficient to support ATP hydrolysis and translocation in an ABC transporter, the histidine permease. J Biol Chem 274, 26727–26735.[CrossRef]
    [Google Scholar]
  23. Pollet, J.-F., Geffel, J. V., Stevens, E. V., Geffel, R. V., Beauwens, R., Bollen, A. & Jacobs, P. ( 2000; ). Expression and intracellular processing of chimeric and mutant CFTR molecules. Biochim Biophys Acta 1500, 59–69.[CrossRef]
    [Google Scholar]
  24. Prasad, R., Worgifosse, P. D., Goffeau, A. & Balzi, E. ( 1995; ). Molecular cloning and characterisation of a novel gene of C. albicans, CDR1, conferring multiple resistance to drugs and antifungals. Curr Genet 27, 320–329.[CrossRef]
    [Google Scholar]
  25. Prasad, R., Krishnamurthy, S., Gupta, V. & Panwar, S. L. ( 1998; ). Multidrug transporters of Candida albicans. Folia Microbiol 43, 228.
    [Google Scholar]
  26. Rice, P., Longden, I. & Bleasby, A. ( 2000; ). EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16, 276–277.[CrossRef]
    [Google Scholar]
  27. Ruetz, S. & Gros, P. ( 1994; ). Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell 77, 1071–1081.[CrossRef]
    [Google Scholar]
  28. Sanglard, D., Kuchler, K., Ischer, F., Pagani, J.-L., Monod, M. & Bille, J. ( 1995; ). Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 39, 2378–2386.[CrossRef]
    [Google Scholar]
  29. Sanglard, D., Ischer, F., Monod, M. & Bille, J. ( 1997; ). Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene. Microbiology 143, 405–416.[CrossRef]
    [Google Scholar]
  30. Sanglard, D., Ischer, F., Monod, M., Dogra, S., Prasad, R. & Bille, J. ( 1999; ). Analysis of the ATP-binding cassette (ABC)-transporter gene CDR4 from Candida albicans. In ASM Conference on Candida and Candidiasis, Charleston, SC, USA, March 1–4, p. 56.
  31. Shukla, S., Saini, P., Smriti, Jha, S., Ambudkar, S. V. & Prasad, R. ( 2003; ). Functional characterization of Candida albicans ABC transporter Cdr1p. Eukaryot Cell 2, 1361–1375.[CrossRef]
    [Google Scholar]
  32. 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.[CrossRef]
    [Google Scholar]
  33. Smit, J. J., Schinkel, A. H., Oude Elferink, R. P. J. & 11 other authors ( 1993; ). Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to complete absence of phospholipid from bile and to liver disease. Cell 75, 451–462.[CrossRef]
    [Google Scholar]
  34. Smriti, Krishnamurthy, S., Dixit, B. L., Gupta, C. M., Milewski, S. & Prasad, R. ( 2002; ). ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators. Yeast 19, 303–318.[CrossRef]
    [Google Scholar]
  35. Tusnady, G. E. & Simon, I. ( 1998; ). Principles governing amino acid composition of integral membrane proteins: applications to topology prediction. J Mol Biol 283, 489–506.[CrossRef]
    [Google Scholar]
  36. Tusnady, G. E. & Simon, I. ( 2001; ). The HMMTOP transmembrane topology prediction server. Bioinformatics 17, 849–850.[CrossRef]
    [Google Scholar]
  37. Walmsley, M. B., Mckeegan, K. S. & Walmsley, A. R. ( 2003; ). Structure and function in efflux pumps that confer resistance to drugs. Biochem J 376, 313–338.[CrossRef]
    [Google Scholar]
  38. White, T. C., Marr, K. A. & Bowden, R. A. ( 1998; ). Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11, 382–402.
    [Google Scholar]
  39. Zhou, Y., Gottesman, M. M. & Pastan, I. ( 1999; ). Domain exchangeability between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2). Mol Pharmacol 56, 997–1004.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28471-0
Loading
/content/journal/micro/10.1099/mic.0.28471-0
Loading

Data & Media loading...

Supplements

vol. , part 5, pp. 1559 - 1573

Amino acid sequence alignment of Cdr1p and Cdr3p. [ PDF] (55 kb)



PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error