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Overexpression of multidrug efflux pumps often creates broad\u2010spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild\u2010type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3\u20134x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild\u2010type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.<\/jats:p>","DOI":"10.1111\/mmi.14351","type":"journal-article","created":{"date-parts":[[2019,7,11]],"date-time":"2019-07-11T13:47:40Z","timestamp":1562852860000},"page":"1131-1144","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites"],"prefix":"10.1111","volume":"112","author":[{"given":"Nidhi","family":"Arya","sequence":"first","affiliation":[{"name":"The Department of Biology Catholic University of America Washington DC 20064 USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hadiar","family":"Rahman","sequence":"additional","affiliation":[{"name":"The Department of 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