MDR1 is a member of the major facilitator super-family that contributes to multidrug resistance of Candida albicans . This antiporter belongs to the Drug/H+Antiporter 1 (DHA1) family, pairing 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 the proton movement, we explored the role of all charged residues of the twelve transmembrane segments (TMS) of Mdr1p in drug/H+ transport by mutational analysis. This revealed that the conserved residue R215, positioned close to the C-terminal end of TMS-4, is critical for drug/H+ antiport, allowing protonation over a range of pH, contrarily to its H215 or K215 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 drugs docking. The model points to the importance of R215 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 establish not only a new system for monitoring drug/H+ transport but also unveil a positively charged residue critical for Mdr1p function.
- Candida albicans
- ©2016 The Author(s)
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