Transport Mechanism and pH Regulation of the Na+/H+ Antiporter NhaA from Escherichia coli

Mager, Thomas; Rimon, Abraham; Padan, Etana; Fendler, Klaus

doi:10.1074/jbc.m111.230235

Cited by 90 publications

(188 citation statements)

References 39 publications

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“…The presence of a FRET signal at 528 nm in the fusion protein of N39-CFP-C-YFP but not in N39-CFP provided the possibility that the N-and C-terminal regions are structurally close in proximity and synergistically functional in the NhaD antiporters. It is well known that negatively charged residues are important for ion binding or pH sensing in cation exchange antiporters (22,26,27). Prompted by the functional importance of the N terminus in the NhaD antiporter, we compared the sequences of the N-terminal regions in these proteins and found a unique negatively charged residue (Glu 38 ) in NhaD2, which was replaced by Pro in NhaD1.…”

Section: Discussionmentioning

confidence: 99%

Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2

et al. 2017

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Two NhaD-type antiporters, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. strain Y2, exhibit different physiological functions in regard to Na ϩ and Li ϩ resistance, although they share high sequence identity. In the present study, the truncation of an additional 4 C-terminal residues from NhaD2 or an exchange of 39 N-terminal residues between these proteins resulted in the complete loss of antiporter activity. Interestingly, combining 39 N-terminal residues and 7 C-terminal residues of NhaD2 (N39D2-C7) partially recovered the activity for Na ϩ and Li ϩ expulsion, as well as complementary growth following exposure to 300 mM Na ϩ and 100 mM Li ϩ stress. The recovered activity of chimera N39D2-C7 indicated that the N and C termini are structurally dependent on each other and function synergistically. Furthermore, fluorescence resonance energy transfer (FRET) analysis suggested that the N and C termini are relatively close in proximity which may account for their synergistic function in ion translocation. In the N-terminal region of N39D2-C7, the replacement of Glu 38 with Pro abolished the recovered complementary and transport activities. In addition, this amino acid substitution in NhaD2 resulted in a drastically decreased complementation ability in Escherichia coli KNabc (level identical to that of NhaD1), as well as decreased activity and an altered pH profile.IMPORTANCE Limited information on NhaD antiporters supports speculation that these antiporters are important for resistance to high salinity and alkalinity. Moreover, only a few functional residues have been identified in NhaD antiporters, and there is limited literature on the molecular mechanisms of NhaD antiporter activity. The altered antiporter abilities of chimeras and mutants in this study implicate the functions of the N and C termini, especially Glu 38 , in pH regulation and ion translocation, and, most importantly, the essential roles of this negatively charged residue in maintaining the physiological function of NhaD2. These findings further our understanding of the molecular mechanism of NhaD antiporters for ion transport.

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Section: Discussionmentioning

confidence: 99%

Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2

et al. 2017

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“…It is important to note that when bacterial cells are exposed to conditions which require proton influx into the cytoplasm, such as alkaline growth conditions, the expression of proton-pumping F 0 F 1 -ATP synthase is elevated in E. coli along with a repression of proton-extruding respiratory chain complexes (39). Monovalent cations like Na ϩ can stimulate the activity of several classes of Na ϩ /K ϩ proton antiporters, many of which have a central role in pH homeostasis under a variety of growth conditions (for example, NhaA [76,77] and MdfA [42]). However, since both Mg 2ϩ and Na ϩ rely on Cpx activation to rescue the temperature sensitivity of BC202, it is possible that some Cpx-induced gene(s) is required for these conditions to rescue the growth defect of BC202.…”

Section: Discussionmentioning

confidence: 99%

Multiple Envelope Stress Response Pathways Are Activated in an Escherichia coli Strain with Mutations in Two Members of the DedA Membrane Protein Family

2013

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We have reported that simultaneous deletion of two Escherichia coli genes, yqjA and yghB, encoding related and conserved inner membrane proteins belonging to the DedA protein family results in a number of intriguing phenotypes, including temperature sensitivity at 42°C, altered membrane lipid composition, and cell division defects. We sought to characterize these and other phenotypes in an effort to establish a function for this protein family in E. coli. Here, using reporter assays, we show that the major envelope stress response pathways Cpx, Psp, Bae, and Rcs are activated in strain BC202 (W3110; ⌬yqjA ⌬yghB) at the permissive growth temperature of 30°C. We previously demonstrated that 10 mM Mg 2؉ , 400 mM NaCl, and overexpression of tatABC are capable of restoring normal growth to BC202 at elevated growth temperatures. Deletion of the cpxR gene from BC202 results in the loss of the ability of these supplements to restore growth at 42°C. Additionally, we report that the membrane potential of BC202 is significantly reduced and that cell division and growth can be restored either by expression of the multidrug transporter MdfA from a multicopy plasmid or by growth at pH 6.0. Together, these results suggest that the DedA family proteins YqjA and YghB are required for general envelope maintenance and homeostasis of the proton motive force under a variety of growth conditions.

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“…An increase in extracellular Mg 2ϩ can enhance membrane integrity and can also inhibit the activity of the F o /F 1 proton-transporting ATPase (41), which is also upregulated in response to alkaline growth conditions (12). Finally, high levels of monovalent cations such as Na ϩ or K ϩ can stimulate a number of proton-dependent antiporters, such as NhaA and MdfA, which play critical roles in cellular pH homeostasis (16,42). Interestingly, the ability of 400 mM Na or 10 mM Mg to restore growth and cell division to BC202 is dependent upon an intact Cpx envelope stress response pathway, suggesting that some Cpx-induced gene may be required for this effect (10).…”

Section: Discussionmentioning

confidence: 99%

Members of the Conserved DedA Family Are Likely Membrane Transporters and Are Required for Drug Resistance in Escherichia coli

Kumar

Doerrler

2014

Antimicrob Agents Chemother

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Bacterial resistance to antibiotics and biocides is an increasing public health problem. Genes encoding integral membrane proteins belonging to the DedA family are present in most bacterial genomes, including Escherichia coli. An E. coli strain lacking partially redundant DedA family genes yqjA and yghB (strain BC202) displays temperature sensitivity and cell division defects. These phenotypes can be corrected by overexpression of mdfA, an Na ؉ -K ؉ /H ؉ antiporter of the major facilitator superfamily. We show that BC202 is hypersensitive to several biocides and cationic compounds that are known substrates of several multidrug resistance transporters, including MdfA, EmrE, and AcrB. The introduction of deletions of genes encoding these drug transporters into BC202 results in additional sensitivity. Expression of wild-type yghB or yqjA can restore drug resistance, but this is eliminated upon mutation of two membrane-embedded acidic amino acids (E39 or D51 in either protein). This dependence upon membrane-embedded acidic amino acids is a hallmark of proton-dependent antiporters. Overexpression of mdfA in BC202 or artificially restoring proton motive force (PMF) restores wild-type resistance to substrates of MdfA as well as other drug resistance transporters such as EmrE and AcrAB. These results suggest that YqjA and YghB may be membrane transporters required for PMF-dependent drug efflux in E. coli.

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Transport Mechanism and pH Regulation of the Na+/H+ Antiporter NhaA from Escherichia coli

Cited by 90 publications

References 39 publications

Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2

Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2

Multiple Envelope Stress Response Pathways Are Activated in an Escherichia coli Strain with Mutations in Two Members of the DedA Membrane Protein Family

Members of the Conserved DedA Family Are Likely Membrane Transporters and Are Required for Drug Resistance in Escherichia coli

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