Search

A truncated human Na+/glucose cotransporter (C5, residues 407–664) was expressed and purified from Escherichia coliusing a GST fusion vector and glutathione affinity chromatography. The truncated transporter (C5) was cleaved from GST‐C5by Factor Xa proteolysis and purified by gel filtration chromatography. Up to 1 mg of purified GST‐C5was obtained from 1 l bacterial culture. Reconstitution of both GST‐C5and C5proteins into lipid vesicles resulted in 2.5‐fold higher initial uptake rates of [3H]D‐glucose into C5‐proteoliposomes than into liposomes. Transport was stereospecific, saturable, and inhibited by phloretin. These properties are similar to those obtained for C5in Xenopus laevisoocytes, and provide additional evidence that the five C‐terminal transmembrane helices in SGLT1 form the sugar translocation pathway.

To investigate the role of charged intramembrane residues in the function of the rabbit Na +/glucose cotransporter (rbSGLT1) we substituted arginine-427 (R427) by alanine in the putative domain M9 SGLT1. This residue is conserved in all the members of the SGLT1 family. The mutant protein (R427A) was expressed in Xenopusoocytes and, although Western blot analysis revealed that it was produced in amounts comparable to wildtype, no function was measured. Freeze-fracture analysis showed that R427A SGLT1 was not in the plasma membrane while immunocytochemical experiments localized the transporter to just beneath it. These results indicate that arginine-427 plays a critical role in SGLT1 trafficking to the plasma membrane.