Characterization of the Vibrio parahaemolyticus Na+/Glucose cotransporter. A bacterial member of the sodium/glucose transporter (SGLT) family.
The Vibrio parahaemolyticus sodium/glucose transporter (vSGLT) is a bacterial member of the SGLT gene family. Wild-type and mutant vSGLT proteins were expressed in Escherichia coli, and transport activity was measured in intact cells and plasma membrane vesicles. Two cysteine-less vSGLT proteins exhibited sugar transport rates comparable with that of the wild-type protein. Six residues in two regions of vSGLT known to be of functional importance in SGLT1 were replaced individually with cysteine in the cysteine-less protein. Characterization of these single cysteine-substituted vSGLTs showed that two residues (Gly-151 and Gln-428) are essential for transport function, whereas the other four residues (Leu-147, Leu-149, Ala-423, and Gln-425) are not. 2-Aminoethylmethanethiosulfonate (MTSEA) blocked Na(+)/glucose transport by only the transporter bearing a cysteine at position 425 (Q425C). MTSEA inhibition was reversed by dithiothreitol and blocked by the presence of both Na(+) and d-glucose, indicating that conformational changes of the vSGLT protein are involved in Na(+)/glucose transport. A split version of vSGLT was generated by co-expression of the N-terminal (N(7)) and C-terminal (C(7)) halves of the transporter. The split vSGLT maintained Na(+)-dependent glucose transport activity. Chemical cross-linking of split vSGLT, with a cysteine in each N(7) and C(7) fragment, suggested that hydrophilic loops between helices 4 and 5 and between helices 10 and 11 are within 8 A of each other. We conclude that the mechanism of Na(+)/glucose transport by vSGLT is similar to mammalian SGLTs and that further studies on vSGLT will provide novel insight to the structure and function of this class of cotransporters.
Xie
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Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751, USA.
Turk
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Wright
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D001426:Bacterial Proteins; D001693:Biological Transport, Active; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives; D007202:Indicators and Reagents; D008024:Ligands; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051247:Sodium-Glucose Transport Proteins; D014736:Vibrio parahaemolyticus / Q000378:metabolism*
D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D051247:Sodium-Glucose Transport Proteins; C093171:methanethiosulfonate ethylammonium; C099811:sodium glucose symporter, Vibrio parahaemolyticus; D005020:Ethyl Methanesulfonate