Dissection of Mechanistic Principles of a Secondary Multidrug Efflux Protein

Fluman, Nir; Ryan, Christopher M.; Whitelegge, Julian P.; Bibi, Eitan

doi:10.1016/j.molcel.2012.06.018

Cited by 98 publications

(146 citation statements)

References 35 publications

(73 reference statements)

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“…MdfA from E. coli serves as a model for secondary Mdr transporters of the MFS (7). This transporter exchanges one proton for one monocationic drug molecule, and the protonatable residue in MdfA has recently been identified (D34) (16). This study also revealed that E26 is essential for D34 deprotonation during the antiport cycle.…”

Section: Discussionmentioning

confidence: 71%

“…The hypothetical 3D model of MdfA (12) reveals two membrane-embedded negative charges, Glu26 and Asp34 (Fig. 1), which were previously found to be essential for transport of monovalent cationic substrates (13)(14)(15) and interaction with protons (16). Interestingly, unlike several other Mdr transporters, such as LmrP (17), QacA (18), or EmrE (19), the substrate profile of MdfA does not include divalent cations.…”

mentioning

confidence: 92%

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Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA

Tirosh

Sigal

Gelman

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Multidrug transporters are integral membrane proteins that use cellular energy to actively extrude antibiotics and other toxic compounds from cells. The multidrug/proton antiporter MdfA from Escherichia coli exchanges monovalent cationic substrates for protons with a stoichiometry of 1, meaning that it translocates only one proton per antiport cycle. This may explain why transport of divalent cationic drugs by MdfA is energetically unfavorable. Remarkably, however, we show that MdfA can be easily converted into a divalent cationic drug/≥2 proton-antiporter, either by random mutagenesis or by rational design. The results suggest that exchange of divalent cationic drugs with two (or more) protons requires an additional acidic residue in the multidrug recognition pocket of MdfA. This outcome further illustrates the exceptional promiscuous capabilities of multidrug transporters.

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Section: Discussionmentioning

confidence: 71%

mentioning

confidence: 92%

Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA

Tirosh

Sigal

Gelman

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Such membrane acidic amino acids are found in many proton-dependent transporters belonging to several different families (14)(15)(16)(17)(18). We have recently shown that DedA proteins require these acidic amino acids for the ability to complement the phenotypes of BC202 described above, suggesting they may represent a new family of proton-dependent transporters (12).…”

mentioning

confidence: 93%

Escherichia coli YqjA, a Member of the Conserved DedA/Tvp38 Membrane Protein Family, Is a Putative Osmosensing Transporter Required for Growth at Alkaline pH

Kumar

Doerrler

2015

J Bacteriol

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The ability to persist and grow under alkaline conditions is an important characteristic of many bacteria. In order to survive at alkaline pH, Escherichia coli must maintain a stable cytoplasmic pH of about 7.6. Membrane cation/proton antiporters play a major role in alkaline pH homeostasis by catalyzing active inward proton transport. The DedA/Tvp38 family is a highly conserved membrane protein family of unknown function present in most sequenced genomes. YqjA and YghB are members of the E. coli DedA family with 62% amino acid identity and partially redundant functions. We have shown that E. coli with ⌬yqjA and ⌬yghB mutations cannot properly maintain the proton motive force (PMF) and is compromised in PMF-dependent drug efflux and other PMF-dependent functions. Furthermore, the functions of YqjA and YghB are dependent upon membrane-embedded acidic amino acids, a hallmark of several families of proton-dependent transporters. Here, we show that the ⌬yqjA mutant (but not ⌬yghB) cannot grow under alkaline conditions (ranging from pH 8.5 to 9.5), unlike the parent E. coli. Overexpression of yqjA restores growth at alkaline pH, but only when more than ϳ100 mM sodium or potassium is present in the growth medium. Increasing the osmotic pressure by the addition of sucrose enhances the ability of YqjA to support growth under alkaline conditions in the presence of low salt concentrations, consistent with YqjA functioning as an osmosensor. We suggest that YqjA possesses proton-dependent transport activity that is stimulated by osmolarity and that it plays a significant role in the survival of E. coli at alkaline pH. IMPORTANCEThe ability to survive under alkaline conditions is important for many species of bacteria. Escherichia coli can grow at pH 5.5 to 9.5 while maintaining a constant cytoplasmic pH of about 7.6. Under alkaline conditions, bacteria rely upon proton-dependent transporters to maintain a constant cytoplasmic pH. The DedA/Tvp38 protein family is a highly conserved but poorly characterized family of membrane proteins. Here, we show that the DedA/Tvp38 protein YqjA is critical for E. coli to survive at pH 8.5 to 9.5. YqjA requires sodium and potassium for this function. At low cation concentrations, osmolytes, including sucrose, can facilitate rescue of E. coli growth by YqjA at high pH. These data are consistent with YqjA functioning as an osmosensing cation-dependent proton transporter. Bacterial alkaline pH tolerance is a key feature of pathogenic, ecological, and industrially important bacteria. Microbes have many naturally occurring alkaline habitats, including the human body (1). Neutralophilic bacteria, including Escherichia coli and Vibrio cholerae, can stay viable in alkaline marine environments and can cause threats to public health (2). They have the ability to maintain their intracellular pH in a range of 7.5 to 7.7 when grown within a wide range of pH values between 5.5 and 9.0. Cytoplasmic pH maintenance is vital for structural integrity and the functions of proteins essential for growth...

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“…MdfA is important for substrate but not proton binding (61). MdfA has a 1:1 H ϩ to substrate stoichiometry, and D34 is suggested to be the proton-binding site, indicating an indirect competition mechanism in which protonation of Glu-6 affects the protonation state of Asp-34.…”

Section: H]mppmentioning

confidence: 97%

Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)

Yaffe

Vergara-Jaque

Shuster

et al. 2014

Journal of Biological Chemistry

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Background: Bacterial homologues of neurotransporters served as structural paradigms for interpretation of the functional data available for their eukaryotic counterparts. Results: We identified and characterized a close bacterial homologue of the rat vesicular monoamine transporter rVMAT2. Conclusion: BbMAT is a multidrug antiporter. Conserved membrane-embedded carboxyls play a role in substrate and proton transport. Significance: Understanding of the bacterial homologue should provide insights into rVMAT2.

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Dissection of Mechanistic Principles of a Secondary Multidrug Efflux Protein

Cited by 98 publications

References 35 publications

Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA

Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA

Escherichia coli YqjA, a Member of the Conserved DedA/Tvp38 Membrane Protein Family, Is a Putative Osmosensing Transporter Required for Growth at Alkaline pH

Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)

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