MOLECULAR CHARACTERIZATION OF CATION- COUPLED TRANSPORTERS: THE H+-COUPLED Mg2+- CITRATE TRANSPORTER, CitM, AND THE Na+/SULFATE COTRANSPORTER, hNaSi-1 APPROVED BY THE SUPERVISORY COMMITTEE ________________________________ Dr. Ana M. Pajor ________________________________ Dr. Steven C. King ________________________________ Dr. Joel P. Gallagher ________________________________ Dr. Luis Reuss ________________________________ Dr. Steven A. Weinman ________________________________ Dean, Graduate School MOLECULAR CHARACTERIZATION OF CATION- COUPLED TRANSPORTERS: THE H+-COUPLED Mg2+- CITRATE TRANSPORTER, CitM, AND THE Na+/SULFATE COTRANSPORTER, hNaSi-1 by Hongyan Li DISSERTATION Presented to the Faculty of THE UNIVERSITY OF TEXAS GRADUATE SCHOOL OF BIOMEDICAL SCIENCES AT GALVESTON in partial fulfillment of the requirements for the degree of Doctor of Philosophy THE UNIVERSITY OF TEXAS MEDICAL BRANCH AT GALVESTON May 2003 © Hongyan Li, May, 2003, All Rights Reserved To my dearest parents and everyone else who ever gave me great help and spirit support during my five-year life in Galveston ACKNOWLEDGMENTS I would like to thank the members of my dissertation committee: Ana M. Pajor, Steven C. King, Joel P. Gallagher, Luis Reuss and Steven A. Weinman for their helpful advice and support. Especially, I would like to appreciate my advisor, Ana M. Pajor, for her extreme patience, consideration and encouragement. Also, I would like to thank Steven A. Weinman for his continued encouragement and support. I would like to thank Leoncio Vergara who helped me to finish the beautiful immunofluorescence image of oocytes. Finally, I would like to thank our generous frogs who “donate” their thousands of black and yellow oocytes to the experiments performed in this dissertation. v MOLECULAR CHARACTERIZATION OF CATION- COUPLED TRANSPORTERS: THE H+-COUPLED Mg2+- CITRATE TRANSPORTER, CitM, AND THE Na+/SULFATE COTRANSPORTER, hNaSi-1 Publication No. _______ Hongyan Li M.S. The University of Texas Medical Branch at Galveston, 2003 Supervising Professor Ana M. Pajor In this dissertation, two cation-coupled transporters were characterized at the molecular level. The CitM transporter from Bacillus subtilis was functionally expressed and characterized in E.coli cells. The human NaSi-1 transporter (hNaSi-1) and mutants were functionally expressed in Xenopus oocytes. Antibodies against hNaSi-1 were used to investigate tissue distribution and N-glycosylation. The roles of two conserved serine residues in the transport function of hNaSi-1 were investigated using site-directed mutagenesis and radiotracer assay. CitM belongs to a distinct gene family of secondary active transporters that includes the homologous citrate transporter CitH. In this dissertation, the 2+ Km of CitM for the complex of Mg -citrate was about 300 µM in the presence of saturating Mg2+ concentrations. CitM has a high substrate specificity for citrate. Other tested di- and tricarboxylic acids did not significantly inhibit citrate uptakes in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The transport was inhibited in more alkaline but not in acidic transport buffer and also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin, suggesting a proton-coupled transport. Valinomycin did not affect the uptake vi by CitM, supporting an electroneutral transport model in which one proton is coupled to the uptake of one complex of (Mg2+-citrate)1-. The low affinity Na+/sulfate cotransporter, hNaSi-1, belongs to a specific gene family of Na+-coupled transporters that includes the high affinity hSUT-1 and the Na+-coupled dicarboxylate (NaDC) transporters. Antibodies directed against a peptide of hNaSi-1 recognized the native protein in renal membranes as well as the recombinant protein expressed in Xenopus oocytes. There is a single N-glycosylation site, Asn-591, located at the extracellular C-terminus in hNaSi-1. Site-directed mutagenesis studies of Ser- 260, Ser-288 and the surrounding amino acid residues of hNaSi-1 suggested that these residues are functionally required for hNaSi-1. MTSET inhibition on sulfate uptakes by the four mutants surrounding Ser-260, T257C, T259C, T261C and L263C, was dependent on the cation and substrate used. Since the presence of sodium and sulfate triggers conformational changes during the transport cycle of hNaSi-1, the cation and substrate dependence of MTSET inhibition suggest that these four substituted cysteines move during the transport cycle. Since the four mutated residues are located in TMD-5, this transmembrane domain is also likely to participate in the conformational movement during the transport cycle of hNaSi-1. vii TABLE OF CONTENTS PAGE CHAPTER 1: INTRODUCTION ............................................................................. 1 CELL MEMBRANE TRANSPORT PROTEINS ................................................................... 1 BACTERIAL PROTON-COUPLED TRANSPORTERS ......................................................... 2 2+ PROTON COUPLED Mg -CITRATE TRANSPORTER, CitM............................................ 3 Citrate................................................................................................................... 4 Mg2+-citrate transport system in whole cells and membrane vesicles................. 5 Mg2+-citrate transporter (CitM)........................................................................... 7 Regulation and physiological role of CitM transporter....................................... 9 + MAMMALIAN Na -COUPLED TRANSPORTERS........................................................... 10 + Na -COUPLED SULFATE TRANSPORTER, NaSi-1....................................................... 11 The NaDC/NaSi gene family .............................................................................. 11 Na+-dependent sulfate transporters ................................................................... 12 AIMS OF STUDIES IN THIS DISSERTATION ................................................................. 22 Aim 1: To characterize the function of CitM expressed in E.coli cells.............. 22 Aim 2: To make antibodies against hNaSi-1 and to determine its tissue distribution and N-glycosylation site. ................................................................ 23 Aim 3: To test the hypothesis that the region surrounding Ser-260 and Ser-288 in TMDs 5 and 6 are involved in cation and/or substrate binding by hNaSi-1...... 23 CHAPTER 2: FUNCTIONAL CHARACTERIZATION OF CitM, THE Mg2+- CITRATE TRANSPORTER................................................................................... 30 INTRODUCTION ........................................................................................................ 30 viii METHODS ................................................................................................................ 31 Construction of the CitM expression vector....................................................... 31 Preparation of cells ............................................................................................ 32 Transport assay .................................................................................................. 33 Permeabilization of cells .................................................................................... 35 Protein assay ...................................................................................................... 35 Data analysis...................................................................................................... 36 RESULTS.................................................................................................................. 37 Time course of citrate uptake ............................................................................. 37 Citrate uptakes as a function of citrate concentrations ..................................... 38 Substrate specificity of CitM .............................................................................. 38 Cation effects on citrate uptakes by CitM .......................................................... 39 Effect of pH on citrate uptakes by CitM............................................................. 40 Effect of ionophores on citrate uptakes in CitM................................................. 41 DISCUSSION............................................................................................................. 42 CHAPTER 3: ANTIBODIES AGAINST hNaSi-1 FUSION PROTEIN: TISSUE DISTRIBUTION AND N-GLYCOSYLATION .................................................... 55 INTRODUCTION ........................................................................................................ 55 METHODS ................................................................................................................ 58 Isolation of hNaSi-1 cDNA................................................................................. 58 Site-directed mutagenesis of N-glycosylation site.............................................. 59 Preparation of GST-Si65 fusion protein ............................................................ 60 Preparation and purification of the antibodies.................................................. 62 ix Western blotting ................................................................................................. 62 Preparation of brush border membrane vesicles ............................................... 63 Transcription of cRNA.......................................................................................