tailieunhanh - Báo cáo Y học: Human and Drosophila UDP-galactose transporters transport UDP-N-acetylgalactosamine in addition to UDP-galactose

A putativeDrosophilanucleotide sugar transporter was characterized and shown to be theDrosophilahomologue of the human UDP-Gal transporter (hUGT). When the Drosophila melanogasterUDP-Gal transporter (DmUGT) was expressed in mammalian cells, the transporter protein was localized in the Golgi membranes and complemented the UDP-Gal transport de®ciency of Lec8 cells but not the CMP-Sia transport de®ciency of Lec2 cells. | Eur. J. Biochem. 269 128-138 2002 FEBS 2002 Human and Drosophila UDP-galactose transporters transport UDP-W-acetylgalactosamine in addition to UDP-galactose Hiroaki Segawa Masao Kawakita f and Nobuhiro Ishida Department of Physiological Chemistry The Tokyo Metropolitan Institute of Medical Science Rinshoken Honkomagome Bunkyo-ku Tokyo Japan A putative Drosophila nucleotide sugar transporter was characterized and shown to be the Drosophila homologue of the human UDP-Gal transporter hUGT . When the Drosophila melanogaster UDP-Gal transporter DmUGT was expressed in mammalian cells the transporter protein was localized in the Golgi membranes and complemented the UDP-Gal transport defciency of Lec8 cells but not the CMP-Sia transport deficiency of Lec2 cells. DmUGT and hUGT were expressed in Saccharomyces cerevisiae cells in functionally active forms. Using microsomal vesicles isolated from Saccharomyces cerevisiae expressing these transporters we unexpectedly found that both hUGT and DmUGT could transport UDP-GalNAc as well as UDP-Gal. When amino-acid residues that are conserved among human murine fission yeast and Drosophila UGTs but are distinct from corresponding ones conserved among CMP-Sia transporters CSTs were substituted by those found in CST the mutant transporters were still active in transporting UDP-Gal. One of these mutants in which Asn47 was substituted by Ala showed aberrant intracellular distribution with concomitant destabilization of the protein product. However this mutation was suppressed by an Ile51 to Thr second-site mutation. Both residues were localized within the frst transmembrane helix suggesting that the structure of the helix contributes to the stabilization and substrate recognition of the UGT molecule. Keywords UDP-galactose transporter UDP-galactose UDP-N-acetylgalactosamine nucleotide sugar transporter site-directed mutagenesis. Oligosaccharide chains of secretory and membrane-bound glycoproteins and glycolipids play important roles in