2.A.37 The Monovalent Cation:Proton Antiporter-2 (CPA2) Family
The CPA2 family is a moderately large from bacteria, archaea and eukaryotes. Among the functionally well-characterized members of the family are (1) the KefB/KefC K+ efflux proteins of E. coli which may be capable of catalyzing both K+/H+ antiport and K+ uniport, depending on conditions (Bakker et al., 1987; Booth et al., 1996; Munro et al., 1991), (2) the Na+/H+ antiporter of Enterococcus hirae (Waser et al., 1992) and (3) the K+/H+ antiporter of S. cerevisiae. It has been proposed that under normal physiological conditions, these proteins may function by essentially the same mechanism (Reizer et al., 1992).
KefC and KefB of E. coli are responsible for glutathione-gated K+ efflux (Ferguson et al., 1993, 1997). Each of these proteins consists of a transmembrane hydrophobic N-terminal domain, and a less well-conserved C-terminal hydrophilic domain. Each protein interacts with a second protein encoded by genes that overlap the gene encoding the primary transporter. The KefC ancillary protein is YabF while the KefB ancillary protein is YheR. These ancillary proteins stimulate transport activity about 10-fold (Miller et al., 2000). These proteins are important for cell survival during exposure to toxic metabolites, possibly because they can release K+, allowing H+ uptake. Activation of the KefB or KefC K+ efflux system only occurs in the presence of glutathione and a reactive electrophile such as methylglyoxal or N-ethylmaleimide. Formation of the methylglyoxal-glutathione conjugate, S-lactoylglutathione, is catalyzed by glyoxalase I, and S-lactoylglutathione activates KefB and KefC (MacLean et al., 1998). H+ uptake (acidification of the cytoplasm) accompanying or following K+ efflux may serve as a further protective mechanism against electrophile toxicity (Booth et al., 1996; Ferguson et al., 1993, 1997; Stumpe et al., 1996). Inhibition of transport by glutathione was enhanced by NADH (Fujisawa et al., 2007).
Gram negative bacteria are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. Roosild et al. (2010) have elucidated the mechanism of gating through structural and functional analysis of the E. coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that sense both reduced glutathione, which inhibits Kef activity, and glutathione adducts that form during electrophile detoxification and activate Kef. Roosild et al. (2010) found that reduced glutathione stabilizes an interdomain association between two KTN folds, whereas large adducts sterically disrupt this interaction. F441 is identified as the pivotal residue discriminating between reduced glutathione and its conjugates. They demonstrated a major structural change on the binding of an activating ligand to a KTN-domain protein.
The MagA protein of Magnetospirillum sp. strain AMB-1 has been reported to be required for synthesis of bacterial magnetic particles. The magA gene is subject to transcriptional activation by an iron deficiency (Nakamura et al., 1995). However, are more recent report has shown that magA mutants of both Magnetospirillum magneticum AMB-1 and M. gryphiswaldense MSR-1 formed wild-type-like magnetosomes without a growth defect (Uebe et al. 2012). Its transport function is not known. The GerN and GrmA proteins of Bacillus cereus and Bacillus megaterium, respectively, are spore germination proteins that can exchange Na+ for H+ and/or K+ (Southworth et al., 2001). The AmhT homologue of Bacillus pseudofirmus transports both K+ and NH4+, influences ammonium homeostasis, and is required for normal sporulation and germination. The identification of these proteins as members of the CPA2 family reveals that monovalent cation transport is required for Bacillus spore formation and germination (Tani et al., 1996).
The proteins of the CPA2 family consist of between 333 and 900 amino acyl residues. They exhibit 10-14 transmembrane α-helical spanners (TMSs). Several organisms possess multiple CPA2 paralogues. Thus, E. coli has three, Methanococcus jannaschii has four and Synechocystis sp. has five paralogues. The potassium efflux system, Kef, protects bacteria against the
detrimental effects of electrophilic compounds via acidification of the
cytoplasm. Kef is inhibited by glutathione (GSH) but activated by
glutathione-S-conjugates (GS-X) formed in the presence of electrophiles.
GSH and GS-X bind to overlapping sites on Kef, which are located in a
cytosolic regulatory domain (Healy et al. 2014).
Interestingly, this family proliferated in plants such as Araidopsis thaliana. but is rarely found in metazoans (Bock et al. 2006). These plant proteins probably arose from their bacterial homologues (Chanroj et al. 2012) and privide functions such as osmotic adjustment and K+ homeostasis during pollen development (Sze et al. 2004). These family members show very similar 3-D folds to CPA1 family members, and this structure is called the NhaA-fold (Czerny et al. 2016). H+ pumps, counter-ion fluxes, and cation/H+ antiporters, including CPA2 family members, are probably interlinked with signaling and membrane trafficking to remodel membranes and cell walls (Sze and Chanroj 2018).
The generalized transport reaction catalyzed by members of the CPA2 family is:
M+ (in) + nH+ (out) ⇌ M+ (out) + nH+ (in).
(The carrier-mediated mode)
Some members may also catalyze:
M+ (in) ⇌ M+ (out).
(The channel-mediated mode)
This family belongs to the CPA Superfamily.
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|MacLean, M.J., L.S. Ness, G.P. Ferguson, and I.R. Booth. (1998). The role of glyoxalase I in the detoxification of methylglyoxal and in the activation of the KefB K+ efflux system in Escherichia coli. Mol. Microbiol. 27: 563-571.|
|Miller, S., L.S. Ness, C.M. Wood, B.C. Fox, and I.R. Booth. (2000). Identification of an ancillary protein, YabF, required for activity of the KefC glutathione-gated potassium efflux system in Escherichia coli. J. Bacteriol. 182: 6536-6540.|
|Miller, S., R.M. Douglas, P. Carter, and I.R. Booth. (1997). Mutations in the glutathione-gated KefC K+ efflux system of Escherichia coli that cause constitutive activation. J. Biol. Chem. 272: 24942-24947.|
|Munro, A.W., G.Y. Ritchie, A.J. Lamb, R.M. Douglas, and I.R. Booth. (1991). The cloning and DNA sequence of the gene for the glutathione-regulated potassium-efflux system KefC of Escherichia coli. Mol. Microbiol. 5: 607-616.|
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|Ramírez, J., O. Ramírez, C. Saldaña, R. Coria, and A. Peña. (1998). A Saccharomyces cerevisiae mutant lacking a K+/H+ exchanger. J. Bacteriol. 180: 5860-5865.|
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|Saier, M.H., Jr., B.H. Eng, S. Fard, J. Garg, D.A. Haggerty, W.J. Hutchinson, D.L. Jack, E.C. Lai, H.J. Liu, D.P. Nusinew, A.M. Omar, S.S. Pao, I.T. Paulsen, J.A. Quan, M. Sliwinski, T.-T. Tseng, S. Wachi, and G.B. Young. (1999). Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim. Biophys. Acta 1422: 1-56.|
|Southworth, T.W., A.A. Guffanti, A. Moir, and T.A. Krulwich. (2001). GerN, an endospore germination protein of Bacillus cereus, is an Na+/H+-K+ antiporter. J. Bacteriol. 183: 5896-5903.|
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|Tsunekawa K., Shijuku T., Hayashimoto M., Kojima Y., Onai K., Morishita M., Ishiura M., Kuroda T., Nakamura T., Kobayashi H., Sato M., Toyooka K., Matsuoka K., Omata T. and Uozumi N. (2009). Identification and characterization of the Na+/H+ antiporter Nhas3 from the thylakoid membrane of Synechocystis sp. PCC 6803. J Biol Chem. 284(24):16513-21.|
|Uebe, R., V. Henn, and D. Schüler. (2012). The MagA protein of Magnetospirilla is not involved in bacterial magnetite biomineralization. J. Bacteriol. 194: 1018-1023.|
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|Wei, Y., J. Liu, Y. Ma, and T.A. Krulwich. (2007). Three putative cation/proton antiporters from the soda lake alkaliphile Alkalimonas amylolytica N10 complement an alkali-sensitive Escherichia coli mutant. Microbiology. 153: 2168-2179.|
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|2.A.37.1.1||Glutathione-regulated K+ efflux protein C, KefC; regulated by ancillary protein KefF (YabF)||Bacteria ||KefCF of E. coli |
KefC (P03819) KefF (P0A754)
CPA2 family member of 627 aas and 12 TMSs
CPA2 member of Simonsiella muelleri
Transmembrane and coiled-coil
domain 3,TMCO3 of 677 aas and 13 TMSs. When mutated causes cornea guttata and anterior polar cataracts. he encoding gene is expressed in the human cornea, lens capsule, and choroid-retinal pigment
epithelium (Chen et al. 2016).
TMCO3 of Homo sapiens
Iron-regulated MagA protein. Deletion mutants of magA show apparently normal magnetosomes and growth in spite of an early report to the contrary (Uebe et al. 2012).
MagA of Magnetospirillum sp. strain AMB-1
Glutathione-regulated K+ efflux protein B, KefB; regulated by ancillary protein KefG (YheR)
KefBG of E. coli
|2.A.37.1.3||The K + efflux pump, KefB (Wei et al., 2007)||Bacteria||KefB of Alkalimonas amylolytica (Q0ZAH7)|
K+ efflux antiporter 1, chloroplastic (AtKEA1). Localizes to the chloroplast inner envelope membrane and is essential for normal osmoregulation (Kunz et al. 2014).
KEA1 of Arabidopsis thaliana
Inner membrane protein YbaL
YbaL of Escherichia coli
K+ efflux - K+:H+ antiporter 3 (AtKEA3) of 776 aas and 13 TMSs. Localizes to the thylakoid membrane of the chloroplast. It modulates pmf partitioning through H+ efflux from the lumen, which is critical for photosynthetic acclimation after transitions from high to low light intensity (Armbruster et al. 2014; Kunz et al. 2014).
KEA3 of Arabidopsis thaliana
Chloroplastic K+ efflux antiporter 2, AtKEA2. Cation preference: K+ > Cs+ > Li+ > Na+ (Aranda-Sicilia et al. 2012). The N-terminal hydrophilic domain show sequence similarity to members of the MPA1-C family (8.A.3.1.2). Localizes to the chloroplast inner envelope membrane and plays a role in chloroplast integrity (Kunz et al. 2014). It is up-regulated under salt stress conditions (Yang et al. 2018).
KEA2 of Arabidopsis thaliana
K+ efflux antiporter 5 (AtKEA5)
KEA5 of Arabidopsis thaliana
Glutathione-regulated potassium-efflux system, KefB of 416 aas and 13 TMSs.
KefB of Helicobacter pylori
|2.A.37.2.1||Na+:H+ antiporter, NapA ||Bacteria ||NapA of Enterococcus hirae |
The Na+/H+-K+ antiporter, GerN (spore germination protein-N) (Southworth et al., 2001).
GerN of Bacillus cereus
|2.A.37.2.3||Spore germination protein, GrmA ||Gram-positive bacteria ||GrmA of Bacillus megaterium |
|2.A.37.2.4||The high-affinity (Km(Na+)=0.7 mM) Na+(Li+):H+ thylakoid membrane antiporter, NhaS3 (essential for growth; promotes Na+ resistance; expressed in the presence of high CO2 concentrations; under circadian control (Tsunekawa et al. 2009).|
NhaS3 of Synechocystis sp. PCC6803
Na+/H+ exchanger of 732 aas. The exchanger is the N-terminal domain, and the C-terminal domain is in the cl00292 superfamily of adenine nucleotide alpha hydrolases. This superfamily
includes N-type ATP PPases, ATP sulphurylases, Universal Stress Response
proteins and electron transfer flavoproteins (ETF). The domain forms a
apha/beta/apha fold which binds to adenosine nucleotides.
NHA homologue of Chlorobium limicola
Putative Na+:H+ antiporter of 388 aas and 10 TMSs, Nha.
Nha of Bdellovibrio bacteriovorus
Na+/H+ antiporter of 386 aas and 13 predicted TMSs, NapA. The 3-d structure is known (PDB# 4BWZ; 4BZ2; 4BZ3). In the NapA structure,
the core and dimerization domains are in different positions to those
seen in the E. coli NhaA, and a negatively charged cavity is open to the
outside. The extracellular cavity allows access to a strictly conserved
aspartate residue thought to coordinate ion binding directly. To alternate access to this ion-binding site, however, requires a
surprisingly large rotation of the core domain, some 20° against the
dimerization interface (Lee et al. 2013). A transmembrane lysine residue is essential for electrogenic transport in this and related Na+/H+ antiporters(Uzdavinys et al. 2017).
NapA of Thermus thermophilus
Bacterial CPA2 homologue
CPA2 homologue of Myxococcus xanthus
Monovalent cation antiporter with CBS domain pair of 577 aas.
CPA protein of Pelobacter carbinolicus
Putative Na+:H+ antiporter of 438 aas and 13 TMSs.
NHA protein of Salinispira pacifica
|2.A.37.4.1||K+:H+ antiporter, Kha1 (YJL094c)||Yeast ||Kha1 of Saccharomyces cerevisiae |
Endosome and prevacuole K+:H+ antiporter, CHX17. Mediates potassium ion and pH homeomeostasis, thereby influencing membrane trafficking (Chanroj et al. 2011).
CHX17 of Arabidopsis thaliana (AAX49545)
|2.A.37.4.3||Plasma membrane K+ uptake transporter, CHX13 (Km ≈ 150μM; expressed in roots) (Zhao et al., 2008)||Viridiplantae|
CHX13 of Arabidopsis thaliana (O22920)
CHX08 cation:H+ antiporter (expressed in pollen; Bock et al., 2006) (most like 2.A.37.4.3; 29% identity)
CHX08 of Arabidopsis thaliana (Q58P71)
|2.A.37.4.5||Cation/H(+) antiporter 23, chloroplastic (Protein CATION/H+ EXCHANGER 23) (AtCHX23)||Plants||CHX23 of Arabidopsis thaliana |
|2.A.37.4.6||Cation/H(+) antiporter 26 (Protein CATION/H+ EXCHANGER 26) (AtCHX26)||Plants||CHX26 of Arabidopsis thaliana |
|2.A.37.4.7||Cation/H(+) antiporter 1 (Protein CATION/H+ EXCHANGER 1) (AtCHX1)||Plants||CHX1 of Arabidopsis thaliana |
Endoplasmic reticular K+/H+ antiporter, CHX20. Maintains pH and K+ homeostasis in guard cells. Plays a critical role
in osmoregulation through the control of stomates opening. Regulates alkaline pH-sensitive growth and influences membrane trafficking (Chanroj et al. 2011).
CHX20 of Arabidopsis thaliana
|2.A.37.5.1||The bidirectional K+/NH4+ transporter, AmhT (ammonium homeostasis transporter) (Fujisawa et al., 2007)||Bacteria||AmhTM of Bacillus pseudoforinus|
AmhT (390 aas) (O50576)
AmhM (167 aas) (O50575)
|2.A.37.5.2||The K +/H+ antiporter, YhaTU (Fujisawa et al., 2007)||Bacteria||YhaTU of Bacillus subtilis |
YhaT (165 aas) (O07535)
YhaU (408 aas) (O07536)