title: Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae.
abstract: The SLC13 transporter family, whose members play key physiological roles in the regulation of fatty acid synthesis, adiposity, insulin resistance, and other processes, catalyzes the transport of Krebs cycle intermediates and sulfate across the plasma membrane of mammalian cells. SLC13 transporters are part of the divalent anion:Na(+) symporter (DASS) family that includes several well-characterized bacterial members. Despite sharing significant sequence similarity, the functional characteristics of DASS family members differ with regard to their substrate and coupling ion dependence. The publication of a high resolution structure of dimer VcINDY, a bacterial DASS family member, provides crucial structural insight into this transporter family. However, marrying this structural insight to the current functional understanding of this family also demands a comprehensive analysis of the transporter's functional properties. To this end, we purified VcINDY, reconstituted it into liposomes, and determined its basic functional characteristics. Our data demonstrate that VcINDY is a high affinity, Na(+)-dependent transporter with a preference for C4- and C5-dicarboxylates. Transport of the model substrate, succinate, is highly pH dependent, consistent with VcINDY strongly preferring the substrate's dianionic form. VcINDY transport is electrogenic with succinate coupled to the transport of three or more Na(+) ions. In contrast to succinate, citrate, bound in the VcINDY crystal structure (in an inward-facing conformation), seems to interact only weakly with the transporter in vitro. These transport properties together provide a functional framework for future experimental and computational examinations of the VcINDY transport mechanism.
authors.affiliation: Membrane Transport Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Christopher
authors.initials: C
authors.lastname: Mulligan
authors.affiliation: Membrane Transport Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Fitzgerald
authors.affiliation: The Helen L. and Martin Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, New York, NY 10016 The Helen L. and Martin Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, New York, NY 10016.
authors.forename: Da-Neng
authors.initials: DN
authors.lastname: Wang
authors.affiliation: Membrane Transport Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 mindellj@ninds.nih.gov.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Mindell
mesh_terms: D029382:Dicarboxylic Acid Transporters / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D012964:Sodium / Q000737:chemistry*; D019802:Succinic Acid / Q000737:chemistry*; D014734:Vibrio cholerae / Q000737:chemistry*
chemical_list: D029382:Dicarboxylic Acid Transporters; D008081:Liposomes; D012964:Sodium; D019802:Succinic Acid