GENETIC SEPARATION OF FK506 SUSCEPTIBILITY AND DRUG TRANSPORT IN THE YEAST PDR5 ATP-BINDING CASSETTE MULTIDRUG-RESISTANCE TRANSPORTER

Overexpression of the yeast Pdr5 ATP-binding cassette transporter lead s to pleiotropic drug resistance to a variety of structurally unrelate d 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 Pd r5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into approp riate drug-sensitive yeast cells followed by phenotypic selection of P dr5 mutants. Selected mutant Pdr5 transporters were analyzed with resp ect to their expression levels, subcellular localization, drug resista nce profiles to cycloheximide, rhodamines, antifungal atoles, steroids , and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 m utant genes identified amino acids important for substrate recognition , drug transport, and specific inhibition of the Pdr5 transporter. Mut ations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hy drophilic loops. At least some point mutations identified appear to in fluence folding of Pdr5, suggesting that the folded structure is a maj or substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specifici ty, but also abolished Pdr5 susceptibility to inhibition by the immuno suppressant FK506. This is the first report of a mutation in a yeast A TP-binding cassette transporter that allows for the functional separat ion of substrate transport and inhibitor susceptibility.