Potassium/Proton Antiport System of Escherichia coli

Radchenko, Martha; Tanaka, Kimihiro; Waditee, Rungaroon; Oshimi, Sawako; Matsuzaki, Yasutomo; Fukuhara, Masahiro; Kobayashi, Hiroshi; Takabe, Teruhiro; Nakamura, Tatsunosuke

doi:10.1074/jbc.m600333200

Cited by 74 publications

(71 citation statements)

References 42 publications

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“…lower at neutral pH and significantly increased at alkaline pH. This type of pHdependency is frequently observed for bacterial cation/H + antiporters that have important roles in alkaline pH homeostasis (Padan et al, 2001;Radchenko et al, 2006). These observations support the previous suggestion that the Pha1 system is required to cope with an alkaline environment (Putnoky et al, 1998).…”

Section: Discussionsupporting

confidence: 81%

“…Protein concentration was measured with the BCA protein assay kit (Pierce) using BSA as a standard. Antiporter activity measurements by acridine orange quenching assay were performed as described by Radchenko et al (2006) with slight modifications. Vesicles (25 mg protein) were suspended in 2 ml assay buffer (10 mM BTP, 5 mM MgCl 2 , 140 mM choline chloride, 1.5 mM acridine orange at pH 7.0-9.0) and 4 mM Tris/lactate was added to initiate respiration.…”

Section: Methodsmentioning

confidence: 99%

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pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti

et al. 2009

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The pha1 gene cluster (pha1A′-G) of Sinorhizobium meliloti has previously been characterized as a necessary component for proper invasion into plant root tissue. It has been suggested to encode a multi-subunit K+/H+ antiporter, since mutations in the pha1 region rendered S. meliloti cells sensitive to K+ and alkali, and because there is high amino acid sequence similarity to previously characterized multi-subunit cation/H+ antiporters (Mrp antiporters). However, the detailed transport properties of the Pha1 system are yet to be determined. Interestingly, most of the Mrp antiporters are highly selective for Na+, unlike the Pha1 system. Here, we report the functional expression of the Pha1 system in Escherichia coli and the measurement of cation/H+ antiport activity. We showed that the Pha1 system is indeed a K+/H+ antiporter with a pH optimum under mildly alkaline conditions. Moreover, we found that the Pha1 system can transport Na+; this was unexpected based on previous phenotypic analyses of pha1 mutants. Furthermore, we demonstrated that the cation selectivity of the Pha1 system was altered when the pH was lowered from the optimum. The downregulation of Na+/H+ and K+/H+ antiport activities upon acidic shift appeared to occur via different processes, which might indicate the presence of distinct mechanisms for the regulation of the K+/H+ and Na+/H+ antiport activities of the Pha1 system.

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Section: Discussionsupporting

confidence: 81%

Section: Methodsmentioning

confidence: 99%

pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti

et al. 2009

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“…The K ϩ /H ϩ antiport capacity of KefFC is consistent with predictions from earlier work by Rosen and colleagues (43) of an E. coli K ϩ /H ϩ antiporter that participates in cytoplasmic pH regulation and has a broad substrate range. It may also account for the residual K ϩ /H ϩ antiport activity observed at high pH in a chaA mutant of E. coli (32), which was a level that exceeds the level expected from the multifunctional MdfA antiporter (34).…”

Section: Discussionmentioning

confidence: 99%

“…This host is deficient in K ϩ /H ϩ as well as Na ϩ (Li ϩ )/H ϩ and Ca 2ϩ /H ϩ antiport (31)(32)(33). Tris⅐HCl was used instead of the Tris-Hepes used in earlier assays (11,12), and higher concentrations of K ϩ were tested in view of recent reports of several bacterial K ϩ /H ϩ antiporters with low affinity for cation (32,34). In assays of the B. subtilis YhaTU system, neither the transporter protein YhaU nor the ancillary protein YhaT exhibited K ϩ /H ϩ antiport activity at pH 8.5 when expressed individually.…”

Section: Cpa2mentioning

confidence: 99%

Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity

Fujisawa

Ito

Krulwich

2007

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

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؉ (K ؉ ) efflux system from alkaliphilic Bacillus pseudofirmus OF4; and YhaTU, a K ؉ efflux system from Bacillus subtilis. Here a K ؉ /H ؉ antiport capacity was demonstrated for YhaTU, AmhMT, and KefFC in membrane vesicles from antiporter-deficient E. coli KNabc. The apparent K m for K ؉ was in the low mM range. The peripheral protein was required for YhaU-and KefC-dependent antiport, whereas both AmhT and AmhMT exhibited antiport. KefFC had the broadest range of substrates, using Rb ؉ ϷK ؉ >Li ؉ >Na ؉ . Glutathione significantly inhibited KefFC-mediated K ؉ /H ؉ antiport in vesicles. The inhibition was enhanced by NADH, which presumably binds to the KTN/RCK domain of KefC. The antiport mechanism accounts for the H ؉ uptake involved in KefFC-mediated electrophile resistance in vivo. Because the physiological substrate of AmhMT in the alkaliphile is NH4 ؉ , the results also imply that AmhMT catalyzes NH4 ؉ /H ؉ antiport, which would prevent net cytoplasmic H ؉ loss during NH4 ؉ efflux.

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“…Moreover, E. coli possesses a multiplicity of K + energy-coupled efflux systems that could also participate in the process. 44 …”

Section: Energetics Of Transportmentioning

confidence: 99%

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

et al. 2015

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Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural, low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE, a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent K m 227 AE 21 nM; V max 49 AE 21 pmol Ni 2+ min À1 mg À1 bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(II) ions. This Ni 2+ transport is not inhibited by similar cations such as Mn 2+ , Zn 2+ , or Co 2+ , but is blocked by Cu 2+ . Analysis of site-directed HupE mutants allowed the identification of several residues (H36, D42, H43, F69, E90, H130, and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein.

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Potassium/Proton Antiport System of Escherichia coli

Cited by 74 publications

References 42 publications

pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti

pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti

Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

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