title: Pyruvate formate-lyase interacts directly with the formate channel FocA to regulate formate translocation.
abstract: 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.
authors.affiliation: Institute of Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Doberenz
authors.affiliation: Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle (Saale), Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Zorn
authors.affiliation: Institute of Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
authors.forename: Dörte
authors.initials: D
authors.lastname: Falke
authors.affiliation: Department of Chemistry, Vanderbilt University, 7330 Stevenson Center, Nashville, TN 37235, USA.
authors.forename: David
authors.initials: D
authors.lastname: Nannemann
authors.affiliation: Institute of Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
authors.forename: Doreen
authors.initials: D
authors.lastname: Hunger
authors.affiliation: Institute of Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
authors.forename: Lydia
authors.initials: L
authors.lastname: Beyer
authors.affiliation: Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle (Saale), Germany.
authors.forename: Christian H
authors.initials: CH
authors.lastname: Ihling
authors.affiliation: Department of Chemistry, Vanderbilt University, 7330 Stevenson Center, Nashville, TN 37235, USA.
authors.forename: Jens
authors.initials: J
authors.lastname: Meiler
authors.affiliation: Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle (Saale), Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
authors.affiliation: Institute of Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany. Electronic address: gary.sawers@mikrobiologie.uni-halle.de.
authors.forename: R Gary
authors.initials: RG
authors.lastname: Sawers
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005561:Formates / Q000378:metabolism*; D047468:Immunoprecipitation; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020798:Two-Hybrid System Techniques
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C088103:FocA protein, E coli; D005561:Formates; D026901:Membrane Transport Proteins; D000123:Acetyltransferases; C022560:formate C-acetyltransferase
keywords: chemical cross-linking; computational docking model; fermentation; formate-nitrite transporter; protein–protein interaction