SUMMARY Multidrug transporters are ubiquitous efflux pumps that provide cells with defense against various toxic compounds. In bacteria, which typically harbor numerous multidrug transporter genes, the majority function as secondary multidrug/proton antiporters. Proton-coupled secondary transport is a fundamental process that is not fully understood, largely owing to the obscure nature of proton-transporter interactions. Here we analyzed the substrate/proton coupling mechanism in MdfA, a model multidrug/ proton antiporter. By measuring the effect of protons on substrate binding and by directly measuring proton binding and release, we show that substrates and protons compete for binding to MdfA. Our studies strongly suggest that competition is an integral feature of secondary multidrug transport. We identified the proton-binding acidic residue and show that, surprisingly, the substrate binds at a different site. Together, the results suggest an interesting mode of indirect competition as a mechanism of multi-drug/proton antiport.

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In all living organisms, ribosomes translating membrane proteins are targeted to membrane translocons early in translation, by the ubiquitous signal recognition particle (SRP) system. In eukaryotes, the SRP Alu domain arrests translation elongation of membrane proteins until targeting is complete. Curiously, however, the Alu domain is lacking in most eubacteria. In this study, by analyzing genome-wide data on translation rates, we identified a potential compensatory mechanism in E. coli that serves to slow down the translation during membrane protein targeting. The underlying mechanism is likely programmed into the coding sequence, where Shine–Dalgarno-like elements trigger elongation pauses at strategic positions during the early stages of translation. We provide experimental evidence that slow translation during targeting and improves membrane protein production fidelity, as it correlates with better folding of overexpressed membrane proteins. Thus, slow elongation is important for membrane protein targeting in E. coli, which utilizes mechanisms different from the eukaryotic one to control the translation speed.DOI: http://dx.doi.org/10.7554/eLife.03440.001

The topologies of α-helical membrane proteins are generally thought to be determined during their cotranslational insertion into the membrane. It is typically assumed that membrane topologies remain static after this process has ended. Recent findings, however, question this static view by suggesting that some parts of, or even the whole protein, can reorient in the membrane on a biologically relevant time scale. Here, we focus on antiparallel homo- or heterodimeric small multidrug resistance proteins and examine whether the individual monomers can undergo reversible topological inversion (flip flop) in the membrane until they are trapped in a fixed orientation by dimerization. By perturbing dimerization using various means, we show that the membrane orientation of a monomer is unaffected by the presence or absence of its dimerization partner. Thus, membrane-inserted monomers attain their final orientations independently of dimerization, suggesting that wholesale topological inversion is an unlikely event in vivo.

The largest family of solute transporters includes ion motive force-driven secondary transporters. Several well characterized solute-specific transport systems in this group have at least one irreplaceable acidic residue that plays a critical role in energy coupling during transport. Previous studies have established the importance of acidic residues in substrate recognition by major facilitator superfamily secondary multidrug transporters, but their role in the transport mechanism remained unknown. We have been investigating the involvement of acidic residues in the mechanism of MdfA, an Escherichia coli secondary multidrug/ proton antiporter. We demonstrated that no single negatively charged side chain plays an irreplaceable role in MdfA. Accordingly, we hypothesized that MdfA might be able to utilize at least two acidic residues alternatively. In this study, we present evidence that indeed, unlike solute-specific secondary transporters, MdfA tolerates displacements of an essential negative charge to various locations in the putative drug translocation pathway. The results suggest that MdfA utilizes a proton translocation strategy that is less sensitive to perturbations in the geometry of the proton-binding site, further illustrating the exceptional structural promiscuity of multidrug transporters.