Molecular characterization of Vibrio parahaemolyticus vSGLT - A model for sodium-coupled sugar cotransporters

The Na+/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni2+ affinity chromatography and gel filtration. Resequencing the vSGLT ge ne identified an important correction: the N terminus constitutes an additi onal 13 functionally essential residues. The mass of His-tagged vSGLT expre ssed under its native promoter, as determined by electrospray ionization-ma ss spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of p urified vSGLT yields proteoliposomes active in Na+-dependent galactose upta ke, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 st oichiometry and apparent K-m values of 129 mM (Na+) and 158 mu M (galactose ). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monom er. We conclude that vSGLT is a suitable model for the study of sugar cotra nsporter mechanisms and structure, with potential applicability to the larg er SGLT family of important sodium:solute cotransporters, It is further dem onstrated that ESI-MS is a powerful tool for the study of proteomics of mem brane transporters.