Eukaryotic Cell
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Eukaryotic Cell, July 2007, p. 1150-1165, Vol. 6, No. 7
1535-9778/07/$08.00+0     doi:10.1128/EC.00091-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Three Classes of Membrane Proteins Involved in Fungal Azole Resistance by Functional Hyperexpression in Saccharomyces cerevisiae{triangledown}

Erwin Lamping,1 Brian C. Monk,1 Kyoko Niimi,1 Ann R. Holmes,1 Sarah Tsao,1,{dagger} Koichi Tanabe,2 Masakazu Niimi,2 Yoshimasa Uehara,2 and Richard D. Cannon1*

Department of Oral Sciences, University of Otago, Dunedin, New Zealand,1 Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan2

Received 20 March 2007/ Accepted 10 May 2007

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 hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression 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{Delta}. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, 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 apparent Km value and the Vmax value 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 specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.

* Corresponding author. Mailing address: Department of Oral Sciences, University of Otago, P.O. Box 647, Dunedin 9054, New Zealand. Phone: 64 3 479 7081. Fax: 64 3 479 7078. E-mail: richard.cannon{at}otago.ac.nz

{triangledown} Published ahead of print on 18 May 2007.

{dagger} Present address: Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal, Québec, Canada.

Eukaryotic Cell, July 2007, p. 1150-1165, Vol. 6, No. 7
1535-9778/07/$08.00+0     doi:10.1128/EC.00091-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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Appl. Environ. Microbiol. Infect. Immun. J. Bacteriol.
Mol. Cell Biol. Microbiol. Mol. Biol. Rev. ALL ASM JOURNALS
Copyright © 2007 by the American Society for Microbiology.