{"id":{"repo_id":"utmb","oai_identifier":"oai:utmb-ir.tdl.org:2152.3/95"},"canonical_url":"https://search.dev.ndltd.org/etd/utmb/oai:utmb-ir.tdl.org:2152.3/95","repository":{"repo_id":"utmb","name":"University of Texas Medical Branch","base_url":"https://utmb-ir.tdl.org/server/oai/request"},"display":{"title":"Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1","abstract":"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. \\r\\n\\r\\n CitM belongs to a distinct gene family of secondary active transporters that includes the homologous citrate transporter CitH. In this dissertation, the Km of CitM for the complex of Mg2+-citrate was about 300 mM 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 by CitM, supporting an electroneutral transport model in which one proton is coupled to the uptake of one complex of (Mg2+-citrate)1-. \\r\\n\\r\\nThe 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. \\r\\n","abstract_html":"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. \\r\\n\\r\\n CitM belongs to a distinct gene family of secondary active transporters that includes the homologous citrate transporter CitH. In this dissertation, the Km of CitM for the complex of Mg2+-citrate was about 300 mM 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 by CitM, supporting an electroneutral transport model in which one proton is coupled to the uptake of one complex of (Mg2+-citrate)1-. \\r\\n\\r\\nThe 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. \\r\\n","abstract_has_math":false,"creators":["Hongyan Li"],"institution":"The University of Texas Medical Branch","degree_name":"PhD","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Ana M. Pajor"],"committee_chairs":[],"committee_members":["Steven C. King","Steven A. Weinman","Luis Reuss","Joel P. Gallagher"],"year":2003,"date_issued":"2003-01-28","date_published":"2003-01-28","updated_at":"2026-07-24T05:50:54Z","subjects":["Western blots","radiotracer uptake assay","PCR","mutagenesis","immunofluorescence","brush border membrane vesicles","biotinylation"],"languages":["eng"],"rights":["Copyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works."],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-04102003-012738"],"render_values":[{"text":"etd-04102003-012738","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2152.3/95","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Ana M. Pajor"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Steven C. King","Steven A. Weinman","Luis Reuss","Joel P. Gallagher"]},{"key":"dc:creator","label":"Author","values":["Hongyan Li"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2011-12-20T16:04:36Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2003-05-21","2011-12-20T16:04:36Z"]},{"key":"dc:date.issued","label":"Date","values":["2003-01-28"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["PhD"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["The University of Texas Medical Branch"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Western blots","radiotracer uptake assay","PCR","mutagenesis","immunofluorescence","brush border membrane vesicles","biotinylation"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-04102003-012738"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/2152.3/95"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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. \\r\\n\\r\\n CitM belongs to a distinct gene family of secondary active transporters that includes the homologous citrate transporter CitH. In this dissertation, the Km of CitM for the complex of Mg2+-citrate was about 300 mM 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 by CitM, supporting an electroneutral transport model in which one proton is coupled to the uptake of one complex of (Mg2+-citrate)1-. \\r\\n\\r\\nThe 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. \\r\\n"]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["electronic"]},{"key":"dc:title","label":"Title","values":["Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1"]}]}],"canonical_facts":{"dc:contributor.advisor":["Ana M. Pajor"],"dc:contributor.committeemember":["Steven C. King","Steven A. Weinman","Luis Reuss","Joel P. Gallagher"],"dc:creator":["Hongyan Li"],"dc:date.accessioned":["2011-12-20T16:04:36Z"],"dc:date.available":["2003-05-21","2011-12-20T16:04:36Z"],"dc:date.issued":["2003-01-28"],"dc:description.abstract":["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. \\r\\n\\r\\n CitM belongs to a distinct gene family of secondary active transporters that includes the homologous citrate transporter CitH. In this dissertation, the Km of CitM for the complex of Mg2+-citrate was about 300 mM 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 by CitM, supporting an electroneutral transport model in which one proton is coupled to the uptake of one complex of (Mg2+-citrate)1-. \\r\\n\\r\\nThe 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. \\r\\n"],"dc:format.medium":["electronic"],"dc:identifier.other":["etd-04102003-012738"],"dc:identifier.uri":["http://hdl.handle.net/2152.3/95"],"dc:language.iso":["eng"],"dc:rights":["Copyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works."],"dc:subject":["Western blots","radiotracer uptake assay","PCR","mutagenesis","immunofluorescence","brush border membrane vesicles","biotinylation"],"dc:title":["Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1"],"thesis:degree_level":["Doctoral"],"thesis:degree_name":["PhD"],"thesis:institution_name":["The University of Texas Medical Branch"]},"updated_at":"2026-07-24T05:50:54Z"}