title: Oligomeric state of the oxalate transporter, OxlT.
abstract: OxlT, the oxalate transporter of Oxalobacter formigenes, was studied to determine its oligomeric state in solution and in the membrane. Three independent approaches were used. First, we used triple-detector (SEC-LS) size exclusion chromatography to analyze purified OxlT in detergent/lipid micelles. These measurements evaluate protein mass in a manner independent of contributions from detergent and lipid; such work shows an average OxlT mass near 47 kDa for detergent-solubilized material, consistent with that expected for monomeric OxlT (46 kDa). A disulfide-linked OxlT mutant was used to verify that it was possible detect dimers under these conditions. A second approach used amino-reactive cross-linkers of varying spacer lengths to study OxlT in detergent/lipid micelles and in natural or artificial membranes, followed by analysis via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These tests, performed under conditions where the presence of dimers can be documented for either of two known dimeric transporters (AdiC or TetL), indicate that OxlT exists as a monomer in the membrane and retains this status upon detergent solubilization. In a final test, we showed that reconstitution of OxlT into lipid vesicles at variable protein/lipid ratios has no effect on the specific activity of subsequent oxalate transport, as the OxlT content varies between 0.027 and 5.4 OxlT monomers/proteoliposome. We conclude that OxlT is a functional monomer in the membrane and in detergent/lipid micelles.
authors.affiliation: Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
authors.forename: Di-Cody
authors.initials: DC
authors.lastname: Kang
authors.forename: Prahnesh A
authors.initials: PA
authors.lastname: Venkataraman
authors.forename: Mark E
authors.initials: ME
authors.lastname: Dumont
authors.forename: Peter C
authors.initials: PC
authors.lastname: Maloney
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D003545:Cysteine / Q000235:genetics; D003902:Detergents / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008970:Molecular Weight; D020624:Oxalobacter formigenes / Q000378:metabolism; D055503:Protein Multimerization
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D026901:Membrane Transport Proteins; C099549:OxlT protein, bacteria; D003545:Cysteine