1.A.16 The Formate-Nitrite Transporter (FNT) Family

FNT family members have been sequenced from Gram-negative and Gram-positive bacteria, archaea and yeast. The prokaryotic proteins of the FNT family probably function in the transport of the structurally related compounds, formate, nitrite and hydrosulfide. Formate, nitrite and hydrosulphide transporters respectively are clustered into two (FocA and FdhC), three (NirC-α, NirC-β and NirC-γ) and one (HSC) subfamilies, plus two (YfdC-α and Yfc-β) of unknown specificity (Mukherjee et al. 2017; see below). Certain positions in the two constriction regions and some residues facing the interior show subfamily-specific conservation.  Anions, as such, seem not to traverse the FNT pore (Atkovska and Hub 2017). Instead, anion binding in the pore is energetically coupled to protonation of a centrally located histidine.The histidine can protonate the permeating anion, thereby enabling its release. Such a mechanism may facilitate both export and import of substrates, with or without proton co-transport (Atkovska and Hub 2017).

FNTs exhibit dual transport functionality: at neutral pH, electrogenic anion currents are detectable, whereas upon acidification, transport of the neutral, protonated monoacid predominates. Physiologically, FNT-mediated proton co-transport is vital when monocarboxylic acid products of energy metabolism, such as l-lactate, are released from the cell. Accordingly, Plasmodium falciparum malaria parasites can be killed by small-molecule inhibitors of PfFNT. The proton relay postulate suggests proton transfer from a highly conserved histidine centrally positioned in the transport path, but the dielectric slide mechanism assumes decreasing acidity of substrates entering the lipophilic vestibules and protonation via the bulk water. Helmstetter et al. 2019 defined the transport mechanism of the FNT from the amoebiasis parasite Entamoeba histolytica, EhFNT, and also showed that BtFdhC from Bacillus thuringiensis is a functional formate transporter. Both FNTs carry a nonprotonatable amide amino acid, asparagine or glutamine, respectively, at the central histidine position. Despite having a nonprotonatable residue, EhFNT displayed the same substrate selectivity for larger monocarboxylates including l-lactate. A low substrate affinity is typical for FNTs, and,  proton motive force-dependent transport is observed for PfFNT harboring a central histidine. These results argue against a proton relay mechanism, indicating that substrate protonation must occur outside of the central histidine region, most likely in the vestibules (Helmstetter et al. 2019).

With the exception of the yeast protein (627 amino acyl residues), all members of the family are of about 250-300 residues in length and exhibit 6-8 putative transmembrane α-helical spanners (TMSs). In one case, that of the E. coli FocA protein, a 6 TMS topology has been established. The yeast protein has a similar apparent topology but has a large C-terminal hydrophilic extension of about 400 residues. Formate export and import by the aquaporin-like pentameric formate channel FocA of E. coli is governed by interaction with pyruvate formate-lyase, the enzyme that generates formate (Pinske and Sawers 2016).

The phylogenetic tree shows clustering according to function and organismal phylogeny. The putative formate efflux transporters (FocA) of bacteria associated with pyruvate-formate lyase (pfl) comprise cluster I; the putative formate uptake permeases (FdhC) of bacteria and archaea associated with formate dehydrogenase comprise cluster II; the nitrite uptake permeases (NirC) of bacteria comprise cluster III, and a yeast protein comprises cluster IV (see Mukherjee et al. 2017.

The energy coupling mechanisms for proteins of the FNT family have not been extensively characterized. HCO2- and NO2- uptakes are probably coupled to H+ symport. HCO2- efflux may be driven by the membrane potential by a uniport mechanism or by H+ antiport. FocA of E. coli catalyzes bidirectional formate transport and may function by a channel-type mechanism (Falke et al., 2010).

FocA homologues transports short-chain acids in bacteria, archaea, fungi, algae and certain eukaryotic parasites. Wang et al. (2009) reported the crystal structure of the E. coli FocA at 2.25 Å resolution. FocA forms a symmetric pentamer, with each protomer consisting of six TMSs. Despite a lack of sequence homology, the overall structure of the FocA protomer closely resembles that of aquaporin, indicating that FocA is a channel rather than a carrier. Structural analysis identified potentially important channel residues, defined the channel path and revealed two constriction sites. Unlike aquaporin, FocA is impermeable to water but allows the passage of formate.

FocA (2.A.44.1.1) may be able to switch its mode of operation from a passive export channel at high external pH to a secondary active formate/H+ importer at low pH. The crystal structure of Salmonella typhimurium FocA at pH 4.0 shows that this switch involves a major rearrangement of the amino termini of individual protomers in the pentameric channel (et al., 2011).The amino-terminal helices open or block transport in a concerted, cooperative action that indicates how FocA is gated in a pH-dependent way. Electrophysiological studies show that the protein acts as a specific formate channel at pH 7.0 and that it closes upon a shift of pH to 5.1.

Phylogenetic analysis of prokaryotic FNT sequences revealed eight different subgroups (Mukherjee et al. 2017). Formate, nitrite and hydrosulphide transporters respectively are clustered into two (FocA and FdhC), three (NirC-alpha, NirC-beta and NirC-gamma) and one (HSC) subfamilies plus two FNT subgroups (YfdC-alpha and YfdC-beta) with unassigned function. Structure-based sequence alignments of individual subfamily members revealed that certain positions in the two constriction regions and some residues facing the interior show subfamily-specific conservation (Mukherjee et al. 2017).

Transmembrane transport of monocarboxylates is conferred by structurally diverse membrane proteins. Bader and Beitz 2020 described the pH dependence of lactic acid/lactate facilitation of an aquaporin (AQP9, TC# 1.A.8.9.14), a monocarboxylate transporter (MCT1, SLC16A1, TC# 2.A.1.13.1), and a formate-nitrite transporter (Plasmodium falciparum FNT, PfFNT, TC# 1.A.16.2.7) in the equilibrium transport state. FNTs exhibit a channel-like structure mimicking the aquaporin-fold, yet act as secondary active transporters. Bader and Beitz 2020 used radiolabeled lactate to monitor uptake via yeast-expressed AQP9, MCT1, and PfFNT for long enough time periods to reach the equilibrium state in which import and export rates are balanced. They confirmed that AQP9 behaved perfectly equilibrative for lactic acid, i.e., the neutral lactic acid molecule enters and passes the channel. MCT1, in turn, actively used the transmembrane proton gradient and acted as a lactate/H+ co-transporter. PfFNT behaved highly similar to the MCT in terms of transport properties, although it did not adhere to the classical alternating access transporter model. Instead, FNT appears to use the proton gradient to neutralize the lactate anion in the protein's vestibule to generate lactic acid in a place that traverses the central hydrophobic transport path. Thus, they proposed to include FNT-type proteins into a more generalized, function-based transporter definition (Bader and Beitz 2020).

The probable transport reactions catalyzed by different members of the FNT family are:

(1) RCO2- or NO2- (out) ⇌ RCO2- or NO2- (in)

(2) HCO2- (in) ⇌ HCO2- (out)

(3) HS- (out) ⇌ HS- (in)

This family belongs to the Major Intrinsic Protein (MIP) Superfamily.



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TC#NameOrganismal TypeExample

Formate uptake/efflux permease, FocA.  It catalyzes bidirectional transport, has a pentameric aquaporin-like (TC# 1.A.8) structure, and may function by a channel-type mechanism (Falke et al., 2009; Wang et al. 2009). The structure at 2.25 Å resolution has been determined (Wang et al., 2009).  The protein is encoded in an operon with pyruvate-formate lyase, PflB.  A pyruvate:formate antiport mechanism has been suggested (Moraes and Reithmeier 2012). The C-terminal 6 aas are required for formate transport, but not for homopentamer formation (Hunger et al. 2017).  The N-terminus of FocA interacts with PflB, and this interaction is essential for optimal formate translocation (Doberenz et al. 2014). In fact, the GREs, TdcE and PflB, interact with the FNT channel protein, probably to control formate translocation by FocA (Falke et al. 2016). The lipophilic constrictions of FocA mainly act as barriers to isolate the central histidine from the aqueous bulk, preventing protonation via proton wires. Thus, an FNT transport model is supported in which the central histidine is uncharged, and weak acid substrate anion protonation occurs in the vestibule regions of the transporter before passing the constrictions (Schmidt and Beitz 2021). An interplay between the conserved pore residues Thr-91 and His-209 controls formate translocation through the FocA channel (Kammel et al. 2022). T91 is essential for formate permeation in both directions; however, it is particularly important to allow anion efflux. H209 is essential for formate uptake by FocA, strongly suggesting that protonation-deprotonation of this residue plays a role in formate uptake. These observations substantiate the premise that efflux and influx of formate by FocA are mechanistically distinct processes that are controlled by the interplay between T91 and H209 (Kammel et al. 2022).


FocA of E. coli (P0AC23)


Probable formate transporter 2 (Formate channel 2), FocB (Andrews et al. 1997).


FocB of Escherichia coli


Formate channel, FocA. Competition of formate by Thr90 from the Ω loop may open the channel (Waight et al., 2010).


FocA of Vibrio cholerae (F9A868)


TC#NameOrganismal TypeExample

Formate-specific channel protein, FdhC of 280 aas (Nölling and Reeve 1997).


FdhC of Methanobacterium thermoformicium


Nitrite uptake porter, NitA (Unkles et al., 1991; 2011)


NitA of Aspergillus (Emericella) nidulans


Probable formate uptake permease (Wood et al., 2003).


FdhC of Methanococcus maripaludis


Nitrite uptake porter of 355 aas, Nar1.


Nar1 of Chlamydomonas reinhardtii (Chlamydomonas smithii)


Nitrite channel transporter, NirC, of 382 aas. Structure/function studies including the x-ray structure of the Salmonella orthologue have been reported (Rycovska-Blume et al. 2015).


NirC of Thermofilum pendens


Nitrite/Nitrate exporter of 476 aas, Nar1 (Cabrera et al. 2014).


Nar1 of Pichia angusta (Yeast) (Hansenula polymorpha)


FNT protein of 313 aas and 6 TMSs that transports L-lactacte (Wiechert et al. 2017).  Trophozoites are inhibited by drugs such as MMVOO7839 (Golldack et al. 2017, Hapuarachchi et al. 2017). It seems to transport lactic acid which allows concentrative uptake (Bader and Beitz 2020). However, it exports lactate from inside the parasite to the surrounding parasitophorous vacuole within the erythrocyte cytosol (Lyu et al. 2021).

PfFNT of Plasmodium falciparum


Formate/nitrite (FNT) transporter of 356 aas and 6 TMSs.

FNT of Entamoeba histolytica


Lactate/formate antiporter, possibly energized by the pmf. It is similar to 1.A.16.2.7, and these two proteims are 74% identical to each other).

Lactate/formate:H+ symporter (release from the cytoplasm) of Plasmodium falciparum


TC#NameOrganismal TypeExample

Nitrite uptake/efflux channel (Jia et al. 2009).


NirC of E. coli (P0AC26)


Uncharacterized transporter YwcJ


YwcJ of Bacillus subtilis


Hydrosulfide (hydrogen sulfide; HS-), Fnt3 (Hsc) channel.  Also probably transports chloride, formate and nitrite. The 3-d crystal structure (2.2Å resolution in the closed state) is known (PDB# 3TE2) (Czyzewski and Wang, 2012). The Fnt3 gene is linked to the asrABC operon encoding the sulfite (SO32-) reductase that gives HS- as the product (Czyzewski and Wang 2012).


Hsc or Fnt3 HS- channel of Clostridium difficile (Q186B7)


Nitrite transporter, NirC, of 268 aas and 6 TMSs (Park et al. 2008).

NirC of Klebsiella oxytoca


Formate channel of 283 aas and 6 TMSs, Fnt or FdhC.  Its function has been veritifed (Helmstetter et al. 2019).

FdhC of Bacillus thuringiensis


TC#NameOrganismal TypeExample

Inner membrane protein, YfdC (310aas; 6 TMSs).  May be involved in surfactant resistance (Nakata et al. 2010).


YfdC of E. coli (P37327)


Putative FNT transporter of 346 aas


FNT transporter of Psychrobacter arcticus


FNT homologue  of 313 aas


FNT homologue of Salinarchaeum sp. Harcht-Bsk1


TC#NameOrganismal TypeExample

FNT homologue of 230 aas


FNT homologue of Acholeplasma palmae


FNT homologue of 213 aas


FNT homologue of Acholeplasma laidlawii