Formate-nitrite Transporter Family Research Articles

FocA and its central role in fine-tuning pH homeostasis of enterobacterial formate metabolism.

During enterobacterial mixed-acid fermentation, formate is generated from pyruvate by the glycyl-radical enzyme pyruvate formate-lyase (PflB). In Escherichia coli, especially at low pH, formate is then disproportionated to CO2 and H2 by the cytoplasmically oriented, membrane-associated formate hydrogenlyase (FHL) complex. If electron acceptors are available, however, formate is oxidized by periplasmically oriented, respiratory formate dehydrogenases. Formate translocation across the cytoplasmic membrane is controlled by the formate channel, FocA, a member of the formate-nitrite transporter (FNT) family of homopentameric anion channels. This review highlights recent advances in our understanding of how FocA helps to maintain intracellular formate and pH homeostasis during fermentation. Efflux and influx of formate/formic acid are distinct processes performed by FocA and both are controlled through protein interaction between FocA's N-terminal domain with PflB. Formic acid efflux by FocA helps to maintain cytoplasmic pH balance during exponential-phase growth. Uptake of formate against the electrochemical gradient (inside negative) is energetically and mechanistically challenging for a fermenting bacterium unless coupled with proton/cation symport. Translocation of formate/formic acid into the cytoplasm necessitates an active FHL complex, whose synthesis also depends on formate. Thus, FocA, FHL and PflB function together to govern formate homeostasis. We explain how FocA achieves efflux of formic acid and propose mechanisms for pH-dependent uptake of formate both with and without proton symport. We propose that FocA displays both channel- and transporter-like behaviour. Whether this translocation behaviour is shared by other members of the FNT family is also discussed.

The soluble cytoplasmic N-terminal domain of the FocA channel gates bidirectional formate translocation.

FocA belongs to the pentameric FNT (formate-nitrite transporter) superfamily of anion channels, translocating formate bidirectionally across the cytoplasmic membrane of Escherichia coli and other microorganisms. While the membrane-integral core of FocA shares considerable amino acid sequence conservation with other FNT family members, the soluble cytoplasmic N-terminal domain does not. To analyze the potential biochemical function of FocA's N-terminal domain in vivo, we constructed truncation derivatives and amino acid-exchange variants, and determined their ability to translocate formate across the membrane of E. coli cells by monitoring intracellular formate levels using a formate-sensitive reporter system. Analysis of strains synthesizing these FocA variants provided insights into formate efflux. Strains lacking the ability to generate formate intracellularly allowed us to determine whether these variants could import formate or its toxic chemical analog hypophosphite. Our findings reveal that the N-terminal domain of FocA is crucial for bidirectional FocA-dependent permeation of formate across the membrane. Moreover, we show that an amino acid sequence motif and secondary structural features of the flexible N-terminal domain are important for formate translocation, and efflux/influx is influenced by pyruvate formate-lyase. The soluble N-terminal domain is, therefore, essential for bidirectional formate translocation by FocA, suggesting a "gate-keeper" function controlling anion accessibility.

Overexpression of Chlamydomonas reinhardtii LCIA (CrLCIA) gene increases growth of Nannochloropsis salina CCMP1776

Most aquatic photosynthetic organisms have developed inorganic carbon-concentrating mechanisms (CCMs) to compensate for the kinetic constraints of CO2 concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which functions in the first steps of carbon fixation. CCMs, which are widespread in various types of algae, evolved independently among algal lineages and accordingly play a fundamental role in algal photosynthesis, metabolism, growth, and biomass production. Nannochloropsis, a marine eustigmatophycean microalga, is a candidate organism for biofuel production due to its high lipid content; however, inorganic carbon (Ci) availability in Nannochloropsis cells is not sufficient for complete carbon fixation due to inherently weak CCM machinery, including a CO2-leaking HCO3− pump. CrLCIA, a member of the formate-nitrite transporter family, functions as a HCO3− transporter in Chlamydomonas reinhardti. Thus, in this study, we overexpressed the CrLCIA gene heterologously in N. salina CCMP1776 in an attempt to reinforce its bicarbonate transport activity. CrLCIA expression in N. salina increased intracellular Ci and carbonic anhydrase (CA) activity, resulting in increased growth (30%) and biomass (2-fold). These results indicate that constitutively expressed CrLCIA leads to increased Ci uptake and CA activity, contributing to high availability of CO2 for photosynthesis under low CO2 conditions. The total fatty acid (FA) per cell mass in the transgenic lines was similar to that of wild-type Nannochloropsis cells, indicating that total FA productivity in the transgenic lines is increased approximately 2-fold. These findings will be useful to improve the CCM function for high biomass and lipid production using genetic modification tools in Nannochloropsis species, contributing to improved biodiesel production.

The intracellular parasite Toxoplasma gondii harbors three druggable FNT-type formate and l-lactate transporters in the plasma membrane

Toxoplasma gondii is a globally prevalent parasitic protist. It is well-known for its ability to infect almost all nucleated vertebrate cells, which is reflected by its unique metabolic architecture. Its fast-growing tachyzoite stage catabolizes glucose via glycolysis to yield l-lactate as a major by-product that must be exported from the cell to prevent toxicity; the underlying mechanism remains to be elucidated, however. Herein, we report three formate-nitrite transporter (FNT)-type monocarboxylate/proton symporters located in the plasma membrane of the T. gondii tachyzoite stage. We observed that all three proteins transport both l-lactate and formate in a pH-dependent manner and are inhibited by 2-hydroxy-chromanones (a class of small synthetic molecules). We also show that these compounds pharmacologically inhibit T. gondii growth. Using a chemical biology approach, we identified the critical residues in the substrate-selectivity region of the parasite transporters that determine differential specificity and sensitivity toward both substrates and inhibitors. Our findings further indicate that substrate specificity in FNT family proteins from T. gondii has evolved such that a functional repurposing of prokaryotic-type transporters helps fulfill a critical metabolic role in a clinically important parasitic protist. In summary, we have identified and characterized the lactate transporters of T. gondii and have shown that compounds blocking the FNTs in this parasite can inhibit its growth, suggesting that these transporters could have utility as potential drug targets.

A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum.

The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is extruded into the external medium. In this study, we show that the human malaria parasite Plasmodium falciparum expresses at its surface a member of the microbial formate-nitrite transporter family (PfFNT), which, when expressed in Xenopus laevis oocytes, transports both formate and lactate. The transport characteristics of PfFNT in oocytes (pH-dependence, inhibitor-sensitivity and kinetics) are similar to those of the transport of lactate and formate across the plasma membrane of mature asexual-stage P. falciparum trophozoites, consistent with PfFNT playing a major role in the efflux of lactate and hence in the energy metabolism of the intraerythrocytic parasite.

Identity of a Plasmodium lactate/H(+) symporter structurally unrelated to human transporters.

Maintenance of a high glycolytic flow rate is critical for the rapid growth and virulence of malarial parasites. The parasites release two moles of lactic acid per mole of glucose as the anaerobic end product. However, the molecular identity of the Plasmodium lactate transporter is unknown. Here we show that a member of the microbial formate-nitrite transporter family, PfFNT, acts as a lactate/proton symporter in Plasmodium falciparum. Besides L-lactate, PfFNT transports physiologically relevant D-lactate, as well as pyruvate, acetate and formate, and is inhibited by the antiplasmodial compounds phloretin, furosemide and cinnamate derivatives, but not by p-chloromercuribenzene sulfonate (pCMBS). Our data on PfFNT monocarboxylate transport are consistent with those obtained with living parasites. Moreover, PfFNT is the only transporter of the plasmodial glycolytic pathway for which structure information is available from crystals of homologous proteins, rendering it amenable to further evaluation as a novel antimalarial drug target.

Functional Characterization of the FNT Family Nitrite Transporter of Marine Picocyanobacteria.

Many of the cyanobacterial species found in marine and saline environments have a gene encoding a putative nitrite transporter of the formate/nitrite transporter (FNT) family. The presumed function of the gene (designated nitM) was confirmed by functional expression of the gene from the coastal marine species Synechococcus sp. strain PCC7002 in the nitrite-transport-less mutant (NA4) of the freshwater cyanobacterium Synechococcus elongatus strain PCC7942. The NitM-mediated nitrite uptake showed an apparent Km (NO2−) of about 8 μM and was not inhibited by nitrate, cyanate or formate. Of the nitM orthologs from the three oceanic cyanobacterial species, which are classified as α-cyanobacteria on the basis of the occurrence of Type 1a RuBisCO, the one from Synechococcus sp. strain CC9605 conferred nitrite uptake activity on NA4, but those from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313 did not. A strongly conserved hydrophilic amino acid sequence was found at the C-termini of the deduced NitM sequences from α-cyanobacteria, with a notable exception of the Synechococcus sp. strain CC9605 NitM protein, which entirely lacked the C-terminal amino acids. The C-terminal sequence was not conserved in the NitM proteins from β-cyanobacteria carrying the Type 1b RuBisCO, including the one from Synechococcus sp. strain PCC7002. Expression of the truncated nitM genes from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313, encoding the proteins lacking the conserved C-terminal region, conferred nitrite uptake activity on the NA4 mutant, indicating that the C-terminal region of α-cyanobacterial NitM proteins inhibits the activity of the transporter.

The formate channel FocA exports the products of mixed-acid fermentation

Formate is a major metabolite in the anaerobic fermentation of glucose by many enterobacteria. It is translocated across cellular membranes by the pentameric ion channel/transporter FocA that, together with the nitrite channel NirC, forms the formate/nitrite transporter (FNT) family of membrane transport proteins. Here we have carried out an electrophysiological analysis of FocA from Salmonella typhimurium to characterize the channel properties and assess its specificity toward formate and other possible permeating ions. Single-channel currents for formate, hypophosphite and nitrite revealed two mechanistically distinct modes of gating that reflect different types of structural rearrangements in the transport channel of each FocA protomer. Moreover, FocA did not conduct cations or divalent anions, but the chloride anion was identified as further transported species, along with acetate, lactate and pyruvate. Formate, acetate and lactate are major end products of anaerobic mixed-acid fermentation, the pathway where FocA is predominantly required, so that this channel is ideally adapted to act as a multifunctional export protein to prevent their intracellular accumulation. Because of the high degree of conservation in the residues forming the transport channel among FNT family members, the flexibility in conducting multiple molecules is most likely a general feature of these proteins.

The nitrite transport protein NirC from Salmonella typhimurium is a nitrite/proton antiporter

In anaerobically grown bacteria, transport of nitrite is catalyzed by an integral membrane protein of the form ate–nitrite transporter family, NirC, which in Salmonella typhimurium plays a critical role in intracellular virulence. We present a functional characterization of the S. typhimurium nitrite transporter StmNirC in native membrane vesicles as well as purified and reconstituted into proteoliposomes. Using an electrophysiological technique based on solid supported membranes, we show nitrite induced translocation of negative charges into proteoliposomes reconstituted with purified StmNirC. These data demonstrate the electrogenicity of StmNirC and its substrate specificity for nitrite. Monitoring changes in ΔpH on everted membrane vesicles containing overexpressed StmNirC using acridine orange as a pH indicator we demonstrate that StmNirC acts as a secondary active transporter. It promotes low affinity transport of nitrite coupled to H+ antiport with a pH independent profile in the pH range from 6 to 8. In addition to nitrite also nitrate is transported by StmNirC, but with reduced flux and complete absence of proton antiport activity. Taken together, these data suggest a bispecific anion selectivity of StmNirC with an ion specific transport mode. This may play a role in regulating nitrite transport under physiological conditions.

Formate–nitrite transporters: Optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives

The formate–nitrite transporter family is composed of integral membrane proteins that possess six to eight α-helical transmembrane domains. Genes encoding these proteins are observed widely in prokaryotic genomes as well as certain groups of lower eukaryotes. Thus far, no structural information is available for this transporter family. Towards this aim, and to provide protein for biophysical studies, overexpression of a prokaryotic (TpNirC, from the hyperthermophilic archaebacterium Thermofilum pendens) and an eukaryotic (AnNitA, from the fungus Aspergillus nidulans) representative was achieved in Escherichia coli and Pichia pastoris hosts, respectively. The proteins were purified to >95% homogeneity yielding quantities sufficient for crystallisation trials and were shown by Circular Dichroism (CD) spectroscopy to have a highly α-helical content as expected from in silico predictions. Preliminary investigations by size exclusion chromatography of the oligomeric state of the purified AnNitA protein suggested that it most likely exists as a tetramer.

Unexpected oligomeric structure of the FocA formate channel ofEscherichia coli: a paradigm for the formate–nitrite transporter family of integral membrane proteins

FocA is a predicted formate channel with a deduced mass of 31 kDa that catalyzes the bidirectional movement of formate across the cytoplasmic membrane of Escherichia coli and is the archetype of the formate-nitrite transporter (FNT) family. Overproduced FocA variants with either an N- or a C-terminal Strep-tag increased formate import into anaerobic E. coli cells as determined by the enhanced activity of a single-copy formate-dependent fdhF::lacZ fusion. Using anti-FocA antibodies, we could show that both FocA variants were integrated into the cytoplasmic membrane. Circular dichroism spectroscopy of purified FocA(Strep-N) revealed a high alpha-helical content of 56% consistent with the predicted six transmembrane helices present in the protein. Analysis of the oligomeric state by blue-native polyacrylamide gel electrophoresis revealed FocA to have an unexpected pentameric quaternary structure. This study reports the first isolation of an FNT family member.

Structure of the formate transporter FocA reveals a pentameric aquaporin-like channel

FocA is a representative member of the formate-nitrite transporter family, which transports short-chain acids in bacteria, archaea, fungi, algae and parasites. The structure and transport mechanism of the formate-nitrite transporter family remain unknown. Here we report the crystal structure of Escherichia coli FocA at 2.25 A resolution. FocA forms a symmetric pentamer, with each protomer consisting of six transmembrane segments. Despite a lack of sequence homology, the overall structure of the FocA protomer closely resembles that of aquaporin and strongly argues that FocA is a channel, rather than a transporter. Structural analysis identifies potentially important channel residues, defines the channel path and reveals two constriction sites. Unlike aquaporin, FocA is impermeable to water but allows the passage of formate. A structural and biochemical investigation provides mechanistic insights into the channel activity of FocA.

Chloride Homeostasis in Saccharomyces cerevisiae: High Affinity Influx, V-ATPase-dependent Sequestration, and Identification of a Candidate Cl− Sensor

Chloride homeostasis in Saccharomyces cerevisiae has been characterized with the goal of identifying new Cl− transport and regulatory pathways. Steady-state cellular Cl− contents (∼0.2 mEq/liter cell water) differ by less than threefold in yeast grown in media containing 0.003–5 mM Cl−. Therefore, yeast have a potent mechanism for maintaining constant cellular Cl− over a wide range of extracellular Cl−. The cell water:medium [Cl−] ratio is >20 in media containing 0.01 mM Cl− and results in part from sequestration of Cl− in organelles, as shown by the effect of deleting genes involved in vacuolar acidification. Organellar sequestration cannot account entirely for the Cl− accumulation, however, because the cell water:medium [Cl−] ratio in low Cl− medium is ∼10 at extracellular pH 4.0 even in vma1 yeast, which lack the vacuolar H+-ATPase. Cellular Cl− accumulation is ATP dependent in both wild type and vma1 strains. The initial 36Cl− influx is a saturable function of extracellular [36Cl−] with K1/2 of 0.02 mM at pH 4.0 and >0.2 mM at pH 7, indicating the presence of a high affinity Cl− transporter in the plasma membrane. The transporter can exchange 36Cl− for either Cl− or Br− far more rapidly than SO4 =, phosphate, formate, HCO3 −, or NO3 −. High affinity Cl− influx is not affected by deletion of any of several genes for possible Cl− transporters. The high affinity Cl− transporter is activated over a period of ∼45 min after shifting cells from high-Cl− to low-Cl− media. Deletion of ORF YHL008c (formate-nitrite transporter family) strongly reduces the rate of activation of the flux. Therefore, Yhl008cp may be part of a Cl−-sensing mechanism that activates the high affinity transporter in a low Cl− medium. This is the first example of a biological system that can regulate cellular Cl− at concentrations far below 1 mM.