Oral Biology pp 219-250 | Cite as

Use of a Yeast-Based Membrane Protein Expression Technology to Overexpress Drug Resistance Efflux Pumps

  • Erwin Lamping
  • Richard D. Cannon
Part of the Methods in Molecular Biology book series (MIMB, volume 666)


Azole antifungal drugs are used widely to treat people with oral fungal infections. Unfortunately, fungi can develop resistance to these drugs. This resistance can be due to the overexpression or mutation of cytochrome P450 14α-lanosterol demethylase, also known as ERG11 or CYP51, and/or the overexpression of membrane-located multidrug efflux pumps. We have developed a heterologous membrane protein expression system that can be used to study the structure and function of these proteins in the non-pathogenic, genetically stable, and versatile eukaryotic model organism, Saccharomyces cerevisiae. In this chapter we describe the techniques used to express the Candida albicans efflux pump Cdr1p in S. cerevisiae.

Key words

Antifungal drug resistance ABC multidrug efflux pumps azole drug target ERG11 CYP51 membrane proteins heterologous expression Saccharomyces cerevisiae 



This work was supported by the Japan Health Sciences Foundation, the NIH, USA (R21DE015075 and R01DE016885 to RDC) and the International Investment Opportunity Fund of the Foundation for Research Science and Technology of New Zealand (UOOX0607).


  1. 1.
    Buckley, M. (2008) The fungal kingdom – diverse and essential roles in earth’s ecosystem. A report based on a colloquium held November 2–4, 2007. American Academy of Microbiology, Washington, DC.Google Scholar
  2. 2.
    Cannon, R. D., Holmes, A. R., Mason, A. B., and Monk, B. C. (1995) Oral Candida: clearance, colonization, or candidiasis? J. Dent. Res, 74, 1152–1161.PubMedCrossRefGoogle Scholar
  3. 3.
    Kelly, S. L., Arnoldi, A., and Kelly, D. E. (1993) Molecular genetic analysis of azole antifungal mode of action. Biochem. Soc. Trans, 21, 1034–1038.PubMedGoogle Scholar
  4. 4.
    Sanglard, D., and Bille, J. (2002) Current understanding of the modes of action of and resistance mechanisms to conventional and emerging antifungal agents for treatment of Candida infections, in Candida and Candidiasis (Calderone, R. A., Ed.). ASM Press, Washington, DC, pp. 349–383.Google Scholar
  5. 5.
    White, T. C., Marr, K. A., and Bowden, R. A. (1998) Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin. Microbiol. Rev, 11, 382–402.PubMedGoogle Scholar
  6. 6.
    Nakamura, K., Niimi, M., Niimi, K., Holmes, A. R., Yates, J. E., Decottignies, A., Monk, B. C., Goffeau, A., and Cannon, R. D. (2001) Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters. Antimicrob. Agents Chemother, 45, 3366–3374.PubMedCrossRefGoogle Scholar
  7. 7.
    Lamping, E., Ranchod, A., Nakamura, K., Tyndall, J. D., Niimi, K., Holmes, A. R., Niimi, M., and Cannon, R. D. (2009) Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei. Antimicrob. Agents Chemother, 53, 354–369.PubMedCrossRefGoogle Scholar
  8. 8.
    Lamping, E., Monk, B. C., Niimi, K., Holmes, A. R., Tsao, S., Tanabe, K., Niimi, M., Uehara, Y., and Cannon, R. D. (2007) Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae. Eukaryot. Cell, 6, 1150–1165.PubMedCrossRefGoogle Scholar
  9. 9.
    Inoue, H., Nojima, H., and Okayama, H. (1990) High efficiency transformation of Escherichia coli with plasmids. Gene, 96, 23–28.PubMedCrossRefGoogle Scholar
  10. 10.
    Schiestl, R. H., and Gietz, R. D. (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet, 16, 339–346.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Erwin Lamping
    • 1
  • Richard D. Cannon
    • 1
  1. 1.Department of Oral Sciences, School of DentistryUniversity of OtagoDunedinNew Zealand

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