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

E. Turk, O. Kim, J. Le Coutre, J. P. Whitelegge, S. Eskandari, J. T. Lam, M. Kreman, G. Zampighi, K. F. Faull, E. M. Wright

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70 Scopus citations

Abstract

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 gene identified an important correction: The N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass 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 purified vSGLT yields proteoliposomes active in Na+-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mM (Na+) and 158 μM (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.

Original languageEnglish (US)
Pages (from-to)25711-25716
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number33
DOIs
StatePublished - Aug 18 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Turk, E., Kim, O., Le Coutre, J., Whitelegge, J. P., Eskandari, S., Lam, J. T., Kreman, M., Zampighi, G., Faull, K. F., & Wright, E. M. (2000). Molecular characterization of Vibrio parahaemolyticus vSGLT. A model for sodium-coupled sugar cotransporters. Journal of Biological Chemistry, 275(33), 25711-25716. https://doi.org/10.1074/jbc.M003127200