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

Research output: Contribution to journalArticle

69 Citations (Scopus)

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

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Vibrio parahaemolyticus
Galactose
Sugars
Electrospray ionization
Electrospray Ionization Mass Spectrometry
Sodium
Mass spectrometry
His-His-His-His-His-His
Affinity chromatography
Fucose
Membrane Transport Proteins
Green Fluorescent Proteins
Affinity Chromatography
Particle Size
Stoichiometry
Proteomics
Electron microscopy
Gel Chromatography
Electron Microscopy
Fusion reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

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

In: Journal of Biological Chemistry, Vol. 275, No. 33, 18.08.2000, p. 25711-25716.

Research output: Contribution to journalArticle

Turk, E, Kim, O, Le Coutre, J, Whitelegge, JP, Eskandari, S, Lam, JT, Kreman, M, Zampighi, G, Faull, KF & Wright, EM 2000, 'Molecular characterization of Vibrio parahaemolyticus vSGLT. A model for sodium-coupled sugar cotransporters', Journal of Biological Chemistry, vol. 275, no. 33, pp. 25711-25716. https://doi.org/10.1074/jbc.M003127200
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. / Molecular characterization of Vibrio parahaemolyticus vSGLT. A model for sodium-coupled sugar cotransporters. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 33. pp. 25711-25716.
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AU - Kim, O.

AU - Le Coutre, J.

AU - Whitelegge, J. P.

AU - Eskandari, S.

AU - Lam, J. T.

AU - Kreman, M.

AU - Zampighi, G.

AU - Faull, K. F.

AU - Wright, E. M.

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