title: Analysis of the binding moiety mediating the interaction between monocarboxylate transporters and carbonic anhydrase II.
abstract: Proton-coupled monocarboxylate transporters (MCTs) mediate the exchange of high energy metabolites like lactate between different cells and tissues. We have reported previously that carbonic anhydrase II augments transport activity of MCT1 and MCT4 by a noncatalytic mechanism, while leaving transport activity of MCT2 unaltered. In the present study, we combined electrophysiological measurements in Xenopus oocytes and pulldown experiments to analyze the direct interaction between carbonic anhydrase II (CAII) and MCT1, MCT2, and MCT4, respectively. Transport activity of MCT2-WT, which lacks a putative CAII-binding site, is not augmented by CAII. However, introduction of a CAII-binding site into the C terminus of MCT2 resulted in CAII-mediated facilitation of MCT2 transport activity. Interestingly, introduction of three glutamic acid residues alone was not sufficient to establish a direct interaction between MCT2 and CAII, but the cluster had to be arranged in a fashion that allowed access to the binding moiety in CAII. We further demonstrate that functional interaction between MCT4 and CAII requires direct binding of the enzyme to the acidic cluster (431)EEE in the C terminus of MCT4 in a similar fashion as previously shown for binding of CAII to the cluster (489)EEE in the C terminus of MCT1. In CAII, binding to MCT1 and MCT4 is mediated by a histidine residue at position 64. Taken together, our results suggest that facilitation of MCT transport activity by CAII requires direct binding between histidine 64 in CAII and a cluster of glutamic acid residues in the C terminus of the transporter that has to be positioned in surroundings that allow access to CAII.
authors.affiliation: From the Department of Biology, Division of Zoology/Membrane Transport and.
authors.forename: Sina Ibne
authors.initials: SI
authors.lastname: Noor
authors.affiliation: From the Department of Biology, Division of Zoology/Membrane Transport and.
authors.forename: Steffen
authors.initials: S
authors.lastname: Dietz
authors.affiliation: From the Department of Biology, Division of Zoology/Membrane Transport and the Department of Biology, Division of General Zoology, University of Kaiserslautern, D-67653 Kaiserslautern, Germany and.
authors.forename: Hella
authors.initials: H
authors.lastname: Heidtmann
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Boone
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610.
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
authors.affiliation: the Department of Biology, Division of General Zoology, University of Kaiserslautern, D-67653 Kaiserslautern, Germany and.
authors.forename: Joachim W
authors.initials: JW
authors.lastname: Deitmer
authors.affiliation: From the Department of Biology, Division of Zoology/Membrane Transport and h.becker@biologie.uni-kl.de.
authors.forename: Holger M
authors.initials: HM
authors.lastname: Becker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D024402:Carbonic Anhydrase II / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D027501:Monocarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis / Q000254:growth & development / Q000378:metabolism
chemical_list: D027501:Monocarboxylic Acid Transporters; D020033:Protein Isoforms; D024402:Carbonic Anhydrase II
keywords: Ion-sensitive Electrode; Lactic Acid; Protein Complex; Protein Expression; Proton Transport; Xenopus; pH Regulation