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.

The C-terminal Six Amino Acids of the FNT Channel FocA Are Required for Formate Translocation But Not Homopentamer Integrity.

FocA is the archetype of the pentameric formate-nitrite transporter (FNT) superfamily of channels, members of which translocate small organic and inorganic anions across the cytoplasmic membrane of microorganisms. The N- and C-termini of each protomer are cytoplasmically oriented. A Y-L-R motif is found immediately after transmembrane helix 6 at the C-terminus of FNT proteins related to FocA, or those with a role in formate translocation. Previous in vivo studies had revealed that formate translocation through FocA was controlled by interaction with the formate-producing glycyl-radical enzyme pyruvate formate-lyase (PflB) or its structural and functional homolog, TdcE. In this study we analyzed the effect on in vivo formate export and import, as well as on the stability of the homopentamer in the membrane, of successively removing amino acid residues from the C-terminus of FocA. Removal of up to five amino acids was without consequence for either formate translocation or oligomer stability. Removal of a sixth residue (R280) prevented formate uptake by FocA in a strain lacking PflB and significantly reduced, but did not prevent, formate export. Sensitivity to the toxic formate analog hypophosphite, which is also transported into the cell by FocA, was also relieved. Circular dichroism spectroscopy and blue-native PAGE analysis revealed, however, that this variant had near identical secondary and quaternary structural properties to those of native FocA. Interaction with the glycyl radical enzyme, TdcE, was also unaffected by removal of the C-terminal 6 amino acid residues, indicating that impaired interaction with TdcE was not the reason for impaired formate translocation. Removal of a further residue (L279) severely restricted formate export, the stability of the protein and its ability to form homopentamers. Together, these studies revealed that the Y278-L279-R280 motif at the C-terminus is essential for bidirectional formate translocation by FocA, but that L279 is both necessary and sufficient for homopentamer integrity.

The glycyl-radical enzyme 2-ketobutyrate formate-lyase, TdcE, interacts specifically with the formate-translocating FNT-channel protein FocA.

Formate is a major product of mixed-acid fermentation in Escherichia coli. Because formate can act as an uncoupler at high concentration it must be excreted from the cell. The FNT (formate-nitrite transporter) membrane channel FocA ensures formate is translocated across the cytoplasmic membrane. Two glycyl-radical enzymes (GREs), pyruvate formate-lyase (PflB) and 2-ketobutyrate formate-lyase (TdcE), generate formate as a product of catalysis during anaerobic growth of Escherichia coli. We demonstrate in this study that TdcE, like PflB, interacts specifically with FocA. His-tagged variants of two other predicted GREs encoded in the genome of E. coli were over-produced and purified and were shown not to interact with FocA, indicating that interaction with FocA is not a general property of GREs per se. Together, these data show that only the GREs TdcE and PflB interact with the FNT channel protein and suggest that, like PflB, TdcE can control formate translocation by FocA.

Pyruvate formate-lyase interacts directly with the formate channel FocA to regulate formate translocation.

The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PflB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA-PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.

Identification of key residues in the formate channel FocA that control import and export of formate.

The formate-nitrite transporter (FNT) family comprises pentameric channels that transport monovalent anions. The prototype of this family is the formate channel (FocA), which was originally identified as a formate channel in Escherichia coli. Each protomer in the channel has a pore with structural features that include periplasmic and cytoplasmic constriction sites, which are likely important for bi-directional gating of substrate passage. Highly conserved amino acid residues within FocA previously identified in structural studies are predicted to be important in the control of formate translocation. Here we present a first detailed in vivo analysis of these residues using a combined targeted amino acid exchange and formate-responsive lacZ fusion-based reporter approach. Sixteen exchanges were made and each variant was shown to be largely unaffected in its secondary and quaternary structure. The invariant H209 and T91 residues, which form part of the lower constriction site linking the Ω-loop with the pore cavity, proved to be important in governing the directionality of formate passage through the pore. A predicted salt-bridge triad of E208-K156-N213 along with the cytoplasmically-oriented N-terminal helix are also involved in pH-dependent gating of the channel. Together, our data are consistent with passive export and import of formate or formic acid through the channel.

Coordination of FocA and pyruvate formate-lyase synthesis in Escherichia coli demonstrates preferential translocation of formate over other mixed-acid fermentation products.

Enterobacteria such as Escherichia coli generate formate, lactate, acetate, and succinate as major acidic fermentation products. Accumulation of these products in the cytoplasm would lead to uncoupling of the membrane potential, and therefore they must be either metabolized rapidly or exported from the cell. E. coli has three membrane-localized formate dehydrogenases (FDHs) that oxidize formate. Two of these have their respective active sites facing the periplasm, and the other is in the cytoplasm. The bidirectional FocA channel translocates formate across the membrane delivering substrate to these FDHs. FocA synthesis is tightly coupled to synthesis of pyruvate formate-lyase (PflB), which generates formate. In this study, we analyze the consequences on the fermentation product spectrum of altering FocA levels, uncoupling FocA from PflB synthesis or blocking formate metabolism. Changing the focA translation initiation codon from GUG to AUG resulted in a 20-fold increase in FocA during fermentation and an ∼3-fold increase in PflB. Nevertheless, the fermentation product spectrum throughout the growth phase remained similar to that of the wild type. Formate, acetate, and succinate were exported, but only formate was reimported by these cells. Lactate accumulated in the growth medium only in mutants lacking FocA, despite retaining active PflB, or when formate could not be metabolized intracellularly. Together, these results indicate that FocA has a strong preference for formate as a substrate in vivo and not other acidic fermentation products. The tight coupling between FocA and PflB synthesis ensures adequate substrate delivery to the appropriate FDH.