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 Naf-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 Angstrom 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.