title: pHluorin enables insights into the transport mechanism of antiporter Mdr1: R215 is critical for drug/H+ antiport.
abstract: Multidrug resistance 1 (MDR1) is a member of the major facilitator superfamily that contributes to MDR of Candida albicans This antiporter belongs to the drug/H(+) antiporter 1 family, pairing the downhill gradient of protons to drug extrusion. Hence, drug efflux from cytosol to extracellular space and the parallel import of H(+) towards cytosol are inextricably linked processes. For monitoring the drug/H(+) antiporter activity of Mdr1p, we developed a new system, exploiting a GFP variant pHluorin, which changes its fluorescence properties with pH. This enabled us to measure the cytosolic pH correlated to drug efflux. Since protonation of charged residues is a key step in proton movement, we explored the role of all charged residues of the 12 transmembrane segments (TMSs) of Mdr1p in drug/H(+) transport by mutational analysis. This revealed that the conserved residue R(215), positioned close to the C-terminal end of TMS-4, is critical for drug/H(+) antiport, allowing protonation over a range of pH, in contrast with its H(215) or K(215) variants that failed to transport drugs at basic pH. Mutation of other residues of TMS-4 highlights the role of this TMS in drug transport, as confirmed by in silico modelling of Mdr1p and docking of drugs. The model points to the importance of R(215) in proton transport, suggesting that it may adopt two main conformations, one oriented towards the extracellular face and the other towards the centre of Mdr1p. Together, our results not only establish a new system for monitoring drug/H(+) transport, but also unveil a positively charged residue critical to Mdr1p function.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Archana Kumari
authors.initials: AK
authors.lastname: Redhu
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Nitesh Kumar
authors.initials: NK
authors.lastname: Khandelwal
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Atanu
authors.initials: A
authors.lastname: Banerjee
authors.affiliation: Drug Resistance & Membrane Proteins Lab, CNRS Research Unit 5086, IBCP, 7 passage du Vercors, F-69007 Lyon, France.
authors.forename: Alexis
authors.initials: A
authors.lastname: Moreno
authors.affiliation: Drug Resistance & Membrane Proteins Lab, CNRS Research Unit 5086, IBCP, 7 passage du Vercors, F-69007 Lyon, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Falson
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Amity Institute of Integrative Sciences and Health, Amity Institute of Biotechnology, Amity University Haryana, Amity Education Valley, Gurgaon 122413, India.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C485187:PHluorin; D049452:Green Fluorescent Proteins
keywords: Candida albicans; Mdr1; antiporter; drug/H+-transport; pHluorin