title: Functional analysis of fungal drug efflux transporters by heterologous expression in Saccharomyces cerevisiae.
abstract: Clinically important resistance of fungal pathogens to azole antifungal drugs is most frequently caused by the over-expression of energy-dependent drug efflux pumps. These pumps usually belong to either the ATP-binding cassette (ABC) family or the Major Facilitator Superfamily (MFS) class of membrane transporter. Little is known about how these pumps work and there is an urgent need to develop pump antagonists that circumvent azole resistance. We have developed a protein hyper-expression system to facilitate functional analysis of efflux pumps based on a Saccharomyces cerevisiae host which has been deleted in seven major ABC transporters to reduce the background of endogenous efflux activity. Plasmid pABC3 was engineered to allow functional hyper-expression of foreign proteins in this host. The main advantages of the system include its ease of directional cloning and the use of homologous recombination to stably integrate single copy constructs into the host genome under the control of a highly active transcriptional regulator. The system has been used to clone and functionally hyper-express genes encoding drug efflux pumps from several pathogenic fungi. Furthermore, the protein hyper-expression system has been used to screen for pump inhibitors and study the structure and function of heterologous membrane proteins.
authors.affiliation: Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo 162-8640, Japan. niimi@nih.go.jp
authors.forename: Masakazu
authors.initials: M
authors.lastname: Niimi
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Wada
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanabe
authors.forename: Aki
authors.initials: A
authors.lastname: Kaneko
authors.forename: Yukie
authors.initials: Y
authors.lastname: Takano
authors.forename: Takashi
authors.initials: T
authors.lastname: Umeyama
authors.forename: Nozomu
authors.initials: N
authors.lastname: Hanaoka
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Uehara
authors.forename: Erwin
authors.initials: E
authors.lastname: Lamping
authors.forename: Kyoko
authors.initials: K
authors.lastname: Niimi
authors.forename: Sarah
authors.initials: S
authors.lastname: Tsao
authors.forename: Ann R
authors.initials: AR
authors.lastname: Holmes
authors.forename: Brian C
authors.initials: BC
authors.lastname: Monk
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cannon
mesh_terms: D000935:Antifungal Agents / Q000378:metabolism*; D025141:Drug Resistance, Fungal / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000935:Antifungal Agents; D026901:Membrane Transport Proteins