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 bacteri al member of the SGLT gene family. Wild-type and mutant vSGLT proteins were expressed in Escherichia coli, and transport activity was measured in inta ct cells and plasma membrane vesicles. Two cysteine less vSGLT proteins exh ibited 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 prot ein. Characterization of these single cysteine-substituted vSGLTs showed th at 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) a re not, 2-Aminoethylmethanethiosulfonate (MTSEA) blocked Na+/glucose transp ort by only the transporter bearing a cysteine at position 425 (Q425C), MTS EA inhibition was reversed by dithiothreitol and blocked by the presence of both Na+ and D-glucose, indicating that conformational changes of the vSGL T protein are involved in Na+/glucose transport. A split version of vSGLT w as 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 hel ices 4 and 5 and between helices 10 and 11 are within 8 Angstrom of each ot her. We conclude that the mechanism of Na+/glucose transport by vSGLT is si milar to mammalian SGLTs and that further studies on vSGLT will provide nov el insight to the structure and function of this class of cotransporters.