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 monovalentanionsacrossbiologicalmembranes.FocA(formatechannelA)translocatesformatebidirectionallybut 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 developedtomonitorchangesinformatelevelsinthecytoplasmrevealedtheimportanceoftheinteractionwithPflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.