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      Functional characterization of a Na +-dependent dicarboxylate transporter from Vibrio cholerae

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          Abstract

          VcINDY, a bacterial homolog of transporters implicated in lifespan in fruit flies and insulin resistance in mammals, is a high affinity, electrogenic, Na +-dependent dicarboxylate transporter.

          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 C 4- and C 5-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.

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          Most cited references48

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          TCDB: the Transporter Classification Database for membrane transport protein analyses and information

          The Transporter Classification Database (TCDB) is a web accessible, curated, relational database containing sequence, classification, structural, functional and evolutionary information about transport systems from a variety of living organisms. TCDB is a curated repository for factual information compiled from >10 000 references, encompassing ∼3000 representative transporters and putative transporters, classified into >400 families. The transporter classification (TC) system is an International Union of Biochemistry and Molecular Biology approved system of nomenclature for transport protein classification. TCDB is freely accessible at . The web interface provides several different methods for accessing the data, including step-by-step access to hierarchical classification, direct search by sequence or TC number and full-text searching. The functional ontology that underlies the database structure facilitates powerful query searches that yield valuable data in a quick and easy way. The TCDB website also offers several tools specifically designed for analyzing the unique characteristics of transport proteins. TCDB not only provides curated information and a tool for classifying newly identified membrane proteins, but also serves as a genome transporter-annotation tool.
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            Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species

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              CITRATE AS AN INTERMEDIARY IN THE INHIBITION OF PHOSPHOFRUCTOKINASE IN RAT HEART MUSCLE BY FATTY ACIDS, KETONE BODIES, PYRUVATE, DIABETES, AND STARVATION.

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                Author and article information

                Journal
                J Gen Physiol
                J. Gen. Physiol
                jgp
                jgp
                The Journal of General Physiology
                The Rockefeller University Press
                0022-1295
                1540-7748
                June 2014
                : 143
                : 6
                : 745-759
                Affiliations
                [1 ]Membrane Transport Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
                [2 ]The Helen L. and Martin Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine and [3 ]Department of Cell Biology, New York University School of Medicine, New York, NY 10016
                Author notes
                Correspondence to Joseph A. Mindell: mindellj@ 123456ninds.nih.gov
                Article
                201311141
                10.1085/jgp.201311141
                4035743
                24821967
                cf721e46-6ef1-4960-afcd-c66e8e959fec
                Copyright @ 2014

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

                History
                : 3 December 2013
                : 21 April 2014
                Categories
                Research Articles

                Anatomy & Physiology
                Anatomy & Physiology

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