ABSTRACT
OxlT, the oxalate transporter of Oxalobacter formigenes, is a member of the Major Facilitator Superfamily of transporters (MFS), one of the largest groups of membrane proteins with substantial relevance to solute transport physiology, pharmacology, and possible drug development. MFS proteins transport a wide range of substrates such as organic and inorganic anions, sugars, drugs, and neurotransmitters. This review succinctly summarizes experimental work on a model MFS protein, OxlT, beginning with its identification as an electrogenic oxalate/formate exchanger, its three-dimensional structure, and discussion of biochemical and biophysical data that have shed further light on its structure and function. We also discuss the structure and function of OxlT in relation to notable MFS carriers such as LacY and GlpT.
Subject(s)
Bacterial Proteins , Membrane Transport Proteins , Models, Biological , Models, Molecular , Oxalobacter formigenes , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biological Transport, Active/physiology , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Oxalic Acid/chemistry , Oxalic Acid/metabolism , Oxalobacter formigenes/chemistry , Oxalobacter formigenes/metabolismABSTRACT
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.
