Functional hyper-expression of membrane proteins in yeast and
discovery of chemosensitizers.
Brian Monk (1), Kyoko Niimi (1), Erwin Lamping (1), Ann Holmes (1), Richard Cannon (1),
David RK Harding (2), Andre Goffeau (3), Anabelle Decottignies
(3), Shun-ici Wada (4), Masakazu Niimi (4), Yoshimasa Uehara (4)
(1) Department of Oral Sciences, University of Otago, Dunedin, New
Zealand;
(2) Centre for Separation Science, Institute for Fundamental Sciences,
Massey University, New Zealand;
(3) FYSA Unit, UCL, Croix du Sud, 2/20, Louvain-la-Neuve, 1348, Louvain-la-Neuve, Belgium (goffeau@fysa.ucl.ac.be);
(4) Department of Bioactive Molecules, National Institute of Infectious
Diseases, Tokyo, Japan
Clinically important resistance to the azole drugs is most frequently
caused by the over-expression of pumps belonging to the ATP-binding
cassette (ABC) family or the Major Facilitator Superfamily (MFS).
Heterologous hyper-expression of these drug resistance determinants from
pathogenic fungi in Sacccharomyces cerevisiae has facilitated
investigation of their structure and function and provided drug
discovery tools. We used the PDR5 promoter and the mutant pdr1-3
transcriptional regulator to drive high level transcription in a S.
cerevisiae host yeast strain deleted of up to seven endogenous ABC
transporters, the functional hyper-expression (>10% of plasma membrane
protein) not only of cloned homologous and heterologous ABC transporters
but also of MFS pumps and the azole target Erg11p ha. Screens using
whole cell chemosensitization to fluconazole has led to the discovery of
novel inhibitors of drug efflux from a 1.8 million member D-octapeptide
combinatorial library. To facilitate the purification and structural
analysis of ABC transporters from secretory vesicles, C-terminal His-tagged ABC transporters have been expressed in the sec6-4
background. The functional hyper-expression of fluconazole resistance
determinants in S. cerevisiae has allowed studies of the
regulation of pump function for the emerging human pathogen Candida
glabrata.