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 Ni(2+) 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 microm (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.
Turk
E
E
Department of Physiology, University of California, Los Angeles 90095- 1751, USA. eturk@mednet.ucla.edu
Kim
O
O
le Coutre
J
J
Whitelegge
J P
JP
Eskandari
S
S
Lam
J T
JT
Kreman
M
M
Zampighi
G
G
Faull
K F
KF
Wright
E M
EM
D001483:Base Sequence; D001692:Biological Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005614:Freeze Fracturing; D005690:Galactose / Q000493:pharmacokinetics; D049452:Green Fluorescent Proteins; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011510:Proteolipids / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D013997:Time Factors; D014736:Vibrio parahaemolyticus / Q000737:chemistry*
D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D051269:Sodium-Glucose Transporter 1; C020485:proteoliposomes; D049452:Green Fluorescent Proteins; D006639:Histidine; D012964:Sodium; D005690:Galactose