title: Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae.
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.
authors.affiliation: Department of Oral Sciences, University of Otago, PO Box 647, Dunedin 9054, New Zealand.
authors.forename: Erwin
authors.initials: E
authors.lastname: Lamping
authors.forename: Brian C
authors.initials: BC
authors.lastname: Monk
authors.forename: Kyoko
authors.initials: K
authors.lastname: Niimi
authors.forename: Ann R
authors.initials: AR
authors.lastname: Holmes
authors.forename: Sarah
authors.initials: S
authors.lastname: Tsao
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanabe
authors.forename: Masakazu
authors.initials: M
authors.lastname: Niimi
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Uehara
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cannon
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000969:Antinematodal Agents / Q000378:metabolism; D001393:Azoles / Q000378:metabolism*; D025141:Drug Resistance, Fungal / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D018942:Macrolides / Q000378:metabolism; D008565:Membrane Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000969:Antinematodal Agents; D001393:Azoles; D018942:Macrolides; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C027837:moxidectin