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Vol. 9, Issue 2, 523-543, February 1998
and
*Department of Molecular Genetics, University and
Biocenter of Vienna, A-1030 Vienna, Austria;
Overexpression of the yeast Pdr5 ATP-binding cassette transporter
leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in
substrate recognition and/or drug transport, we used a combination of
random in vitro mutagenesis and phenotypic screening to isolate novel
mutant Pdr5 transporters with altered substrate specificity. A plasmid
library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by
phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters
were analyzed with respect to their expression levels, subcellular
localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes
identified amino acids important for substrate recognition, drug
transport, and specific inhibition of the Pdr5 transporter. Mutations
were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in
transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the
immunosuppressant FK506. This is the first report of a mutation in a
yeast ATP-binding cassette transporter that allows for the functional
separation of substrate transport and inhibitor susceptibility.
Department
of Biochemistry, Biozentrum, University of Basel, CH-4506 Basel,
Switzerland; and
Institut de Microbiologie, Centre
Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland
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