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

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

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

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

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.

Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae. Publishing Authors By Initials

E Lamping,BC Monk,K Niimi,AR Holmes,S Tsao,K Tanabe,M Niimi,Y Uehara,RD Cannon,

For similar biochemical phenomena, metabolism, and nutrition: biochemical phenomena: substrate specificity research abstracts see: biochemical phenomena, metabolism, and nutrition: biochemical phenomena: substrate specificity research

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

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Eukaryotic cell

VOLUME: 6

Page Numbers: 1150-65

Journal Abbreviation: Eukaryotic Cell

ISSN: 1535-9778

DAY: 18

MONTH: 05

YEAR: 2007

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

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LANGUAGE: eng

NlmUniqueID: 101130731

Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae. Keywords Mesh Terms:

KEYWORDS: Substrate Specificity

MESH TERMS: metabolism

Chemical & Substance for Abstract: Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae. Information

Substance Name: milbemycin

Registry Number: 51570-36-6

Grant and Affiliation Information for Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae.

AFFILIATION: Department of Oral Sciences, University of Otago, PO Box 647, Dunedin 9054, New Zealand.

Country: United States

AGENCY: United States NIDCR

GRANT: R21DE015075-RDC

ACRONYM: DE

MEDLINETA: Eukaryot Cell

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