Eukaryotic Cell doi:10.1128/EC.00091-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Characterization of Three Classes of Membrane Proteins Involved in Fungal Azole Resistance by Functional Hyper-Expression in Saccharomyces cerevisiae
Erwin Lamping,
Brian C. Monk,
Kyoko Niimi,
Ann R. Holmes,
Sarah Tsao,
Koichi Tanabe,
Masakazu Niimi,
Yoshimasa Uehara,
and
Richard D. Cannon*
Department of Oral Sciences, University of Otago, Dunedin, New Zealand, and Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan
* To whom correspondence should be addressed. Email:
richard.cannon{at}otago.ac.nz.
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Abstract |
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The study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyper-expression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p and CneMdr1p, the MFS transporter CaMdr1p and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotype of five pathogenic fungi, and to express human P-glycoprotein (HsAbcb1p). The hyper-expression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomal PDR5 locus of a modified host Saccharomyces cerevisiae strain AD
. Over-expression of heterologous proteins at levels of up to 29% of plasma membrane (PM) protein was achieved. Membrane proteins were expressed with or without GFP-, mRFP-, His-, FLAG/His-, Cys- or His/Cys-tags. Most GFP-tagged proteins tested were correctly trafficked within the cell and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the Kmapp and Vmax of ATPase activities were not significantly affected by the addition of His-tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificity and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains over-expressing ABC drug pumps to fluconazole. The modified hyper-expression system has wide applications for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.
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