Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jul;6(7):1150-65.
doi: 10.1128/EC.00091-07. Epub 2007 May 18.

Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae

Erwin Lamping  1 , Brian C Monk, Kyoko Niimi, Ann R Holmes, Sarah Tsao, Koichi Tanabe, Masakazu Niimi, Yoshimasa Uehara, Richard D Cannon
Affiliations

Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae

Erwin Lamping et al. Eukaryot Cell. 2007 Jul.

Abstract

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, ADDelta. 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 K(m) value and the V(max) 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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
(A) Schematic diagram of the yeast membrane protein hyperexpression system. The system comprises a set of vectors based on pBluescript SK(+), containing a transformation cassette that consists of the PDR5 promoter (dark blue), the PGK1 terminator (green), a selection marker (light blue) (URA3 [pABC3 series of vectors] or HIS1 [pABC5′ series of vectors]), and a small part of the 3′ end of the PDR5 gene (dark blue). Unique 8-bp PacI and NotI cloning sites are located between the PDR5 promoter and the PGK1 terminator for the directional cloning of any ORF. Plasmids pABC3 and pABC5′ provided templates for derivative plasmids that contain a choice of six different C-terminal tags located 3′ to the NotI cloning site (pABC3-tag and pABC5′-tag). The transformation cassette containing an ORF cloned into the PacI and NotI sites can be excised as an AscI fragment and used to transform the host strain AD1-8u−. (B) Confocal microscopy images of AD1-8u− cells overexpressing either the azole drug pump CaCdr1p (i and ii) or the cytosolic protein CaUra3p (iii and iv) tagged with either green (GFP) (i and iii) or red (mRFP) (ii and iv) fluorescent protein. (C) PM proteins (30 μg per lane) of AD1-8u− cells hyperexpressing CaCdr1Ap, CaCdr1Bp, or CaCdr1Ap C-terminally tagged with each of the six tags were separated by SDS-PAGE and visualized with Coomassie blue.
FIG. 2.
FIG. 2.
Strategies for adding His or GFP tags to ORFs. (A) His tags were added by recombinant PCR, followed by transformation of S. cerevisiae strains already containing the heterologous ORF to His+. (B) GFP tags were added by recombinant PCR, followed by transformation of S. cerevisiae strains already containing His tags to Ura+.
FIG. 3.
FIG. 3.
Agarose diffusion assays of AD1-8u− cells overexpressing a selection of fungal ABC drug pumps (ScPdr5p, CaCdr1Ap, CaCdr2Ap, and CneMdr1p), a fungal MFS drug pump, CaMdr1Ap, the azole drug target CaErg11Ap, and the sensitive control strain AD1-8u− containing the empty pABC3 transformation cassette integrated at the chromosomal PDR5 locus (AD/pABC3). Cells were analyzed using agarose diffusion assays as described in Materials and Methods. (A) Susceptibilities to the following xenobiotics were tested: 1, FLC (10 μg); 2, ITC (0.16 μg); 3, MCZ (0.2 μg); 4, KTZ (0.2 μg); 5, NYS (60 μg); 6, R6G (48 μg); 7, cycloheximide (0.2 μg); 8, cerulenin (1 μg); 9, Triton X-100 (1 mg). (B) As in panel A, the substrate specificities of cells overexpressing HsAbcb1p were tested and compared to those of cells overexpressing ScPdr5p using the control strain AD/pABC3. Drugs applied were as follows (top to bottom): 1, FLC (4 μg); 2, ITC (0.5 μg); 3, R6G (80 μg); 4, Triton X-100 (1 mg); 5, aureobasidin A (20 μg).
FIG. 4.
FIG. 4.
SDS-PAGE analysis of PM proteins (30 μg per lane) from AD1-8u− cells overexpressing different fungal and human membrane proteins. In each panel, filled arrowheads indicate heterologous proteins and open arrowheads indicate the endogenous 100-kDa PM H+ ATPase (Pma1p). (A) AD1-8u− cells overexpressing different classes of fungal membrane protein: ABC transporters ScPdr5p and CaCdr1Ap, MFS transporter CaMdr1Ap, and the azole drug target CaErg11Ap, a cytochrome P450 enzyme. pABC3 refers to the drug-sensitive control strain as described in the legend to Fig. 3. (B) The left lane shows 30 μg of PM protein isolated from AD/CaCDR1A-His-p cells. CaCdr1A-His-p after Ni affinity chromatography is shown in the right lane. (C) Fungal drug pumps hyperexpressed in AD1-8u− cells (top panel). The addition of a C-terminal His tag (middle panel) or GFP tag (bottom panel) does not affect their level of expression. Shown below the gel in each panel are the FLC, ITC, and R6G MICs (μg/ml) for each of the overexpressing strains. (D) SDS-PAGE analysis of PM proteins isolated from AD/CaMDR1A, AD/CaERG11A, and the AD/HsABCB1 cells and their GFP-tagged derivatives.
FIG. 5.
FIG. 5.
In vivo pumping activities of different fungal drug pumps using the fluorescent pump substrate R6G. AD1-8u− cells overexpressing drug pumps were grown to mid-logarithmic phase, harvested, and loaded with the fluorescent substrate R6G, and the assay was started by the addition of glucose as an energy source as described in Materials and Methods. The fluorescence intensity accumulating in the supernatant after filtration of the cells was measured and the R6G pumping activity per 107 cells determined. The results are the means ± standard deviations for triplicate determinations.
FIG. 6.
FIG. 6.
Kinetic parameters for the ATPase activities of fungal ABC drug transporters. (A) Vanadate-sensitive ATPase activity (V) versus substrate concentration plot for Cdr1Ap-His/Cys-tagged drug pump at pH 7.5. (B) Lineweaver Burke plot for the vanadate-sensitive ATPase activity of the CaCdr1Ap-His/Cys-tagged drug pump at pH 7.5. The data were taken from panel A. (C) The pH profile for the ATPase activity of untagged CaCdr1Ap (diamond) or CaCdr1Ap with a His tag (square), Cys tag (triangle), or His/Cys tag (cross) is shown. Results are expressed as percentages of activity at pH 7.5.
FIG. 7.
FIG. 7.
Confocal microscopy images of AD1-8u− cells overexpressing different classes of membrane proteins. ABC drug pumps ScPdr5p (a), CaCdr2Ap (c), CgCdr1p (d), and CgPdh1p (e) localized exclusively to the PM, whereas the fungal drug pumps CaCdr1Ap (b) and CkAbc1p (k) also partially localized in internal membrane structures. CneMdr1p (l) and HsAbcb1p (m) were not evenly distributed throughout the PM and were also found in internal structures. The MFS drug pump CaMdr1Ap (o) localized exclusively to the PM, and the azole drug target CaErg11Ap (n) localized to the endoplasmic reticulum, as expected. Light microscopy images of the cells (f to j and p to t) are shown below their confocal images (a to e and k to o).
FIG. 8.
FIG. 8.
Effects of ABC drug pump inhibitors. AD1-8u− cells overexpressing fungal ABC drug pumps ScPdr5p, CaCdr1Ap, CaCdr2Ap, CgCdr1p, CgPdh1p or CkAbc1p, or the control strain overexpressing the MFS drug pump CaMdr1Ap, were analyzed by agarose diffusion assays using CSM solidified with 0.6% agarose and containing either no FLC (left hand plates; control) or FLC at 0.25× MICFLC for each test strain, as described in Materials and Methods. Whatman 3MM paper disks containing the indicated amounts of drug pump inhibitors (1, 5 μg milbemycin α11; 2, 5 μg milbemycin α20; 3, 5 μg milbemycin α25; 4, 5 μg milbemycin β9; 5, 5 μg milbemycin β11; 6, 5 μg enniatin; 7, 25 μg FK506; 8, 50 nmol oligomycin) were placed on each plate.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abbott, A. 2000. Structures by numbers. Nature 408:130-132. - PubMed
    1. Albertson, G. D., M. Niimi, R. D. Cannon, and H. F. Jenkinson. 1996. Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrob. Agents Chemother. 40:2835-2841. - PMC - PubMed
    1. Balzi, E., W. Chen, S. Ulaszewski, E. Capieaux, and A. Goffeau. 1987. The multidrug resistance gene PDR1 from Saccharomyces cerevisiae. J. Biol. Chem. 262:16871-16879. - PubMed
    1. Ben-Yaacov, R., S. Knoller, G. A. Caldwell, J. M. Becker, and Y. Koltin. 1994. Candida albicans gene encoding resistance to benomyl and methotrexate is a multidrug resistance gene. Antimicrob. Agents Chemother. 38:648-652. - PMC - PubMed
    1. Bill, R. M. 2001. Yeast—a panacea for the structure-function analysis of membrane proteins? Curr. Genet. 40:157-171. - PubMed

Publication types

MeSH terms

LinkOut - more resources