Functional reconstitution of SdcS, a Na+-coupled dicarboxylate carrier protein from Staphylococcus aureus.
In Staphylococcus aureus, the transport of dicarboxylates is mediated in part by the Na+-linked carrier protein SdcS. This transporter is a member of the divalent-anion/Na+ symporter (DASS) family, a group that includes the mammalian Na+/dicarboxylate cotransporters NaDC1 and NaDC3. In earlier work, we cloned and expressed SdcS in Escherichia coli and found it to have transport properties similar to those of its eukaryotic counterparts (J. A. Hall and A. M. Pajor, J. Bacteriol. 187:5189-5194, 2005). Here, we report the partial purification and subsequent reconstitution of functional SdcS into liposomes. These proteoliposomes exhibited succinate counterflow activity, as well as Na+ electrochemical-gradient-driven transport. Examination of substrate specificity indicated that the minimal requirement necessary for transport was a four-carbon terminal dicarboxylate backbone and that productive substrate-transporter interaction was sensitive to substitutions at the substrate C-2 and C-3 positions. Further analysis established that SdcS facilitates an electroneutral symport reaction having a 2:1 cation/dicarboxylate ratio. This study represents the first characterization of a reconstituted Na+-coupled DASS family member, thus providing an effective method to evaluate functional, as well as structural, aspects of DASS transporters in a system free of the complexities and constraints associated with native membrane environments.
Hall
Jason A
JA
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0645, USA.
Pajor
Ana M
AM
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D029382:Dicarboxylic Acid Transporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007700:Kinetics; D012964:Sodium / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D029382:Dicarboxylic Acid Transporters; D012964:Sodium