Premium
An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites
Author(s) -
Arya Nidhi,
Rahman Hadiar,
Rudrow Andrew,
Wagner Manuel,
Schmitt Lutz,
Ambudkar Suresh V.,
Golin John
Publication year - 2019
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.14351
Subject(s) - biology , efflux , mutant , rhodamine 123 , wild type , cooperativity , multiple drug resistance , biochemistry , atp binding cassette transporter , transmembrane domain , yeast , quenching (fluorescence) , transporter , biophysics , membrane , fluorescence , gene , antibiotics , physics , quantum mechanics
Summary Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad‐spectrum 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‐type 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 3–4x 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‐type 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.