Substrate recognition and subcellular localization of nucleotide sugar transporters

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Every member of the nucleotide sugar transporter family has distinct and well defined substrate specificity. Molecular chimeras between UDP-Gal transporter (UGT) and CMP-sialic acid (CMP-Sia) transporter (CST) were constructed and analyzed in order to identify submolecular regions responsible for the determination of substrate specificity. Our previous studies indicated that UGT/CST chimeras with their 7th transmembrane helix-containing segment being derived from CST could transport both CMP-Sia and UDP-Gal. In the present study we showed that the N-terminal half of the 7th transmembrane helix of CST is essential for the CMP-Sia transport mediated by the chimeric transporters, and further succeeded in identifying two amino acid residues located in this segment that are particularly important in the recognition of CMP-Sia as a transport substrate.Human UGTrel8 transporter, which is closely related with previously characterized UGTrel7 transporter and Drosophila Frc transporter, was cloned and characterized. Substrate specificity of UGTrel8 was examined using a heterologous expression system in Saccharomyces cerevisiae, and UDP-GlcNAc was identified as its substrate. When expressed in CHO cells, UGTrel8 protein was expressed exclusively in the Golgi membranes in contrast to UGTrel7 which was localized to the endoplasmic reticulum.Frc transporter, a multi-specific UDP-sugar transporter from Drosophila, is specifically required for the synthesis of notch oligosaccharide but not for general glycoconjugate synthesis. The Frc cDNA was transfected into UGT-deficient Lec8 cells to see if Frc clould complement the general carbohydrate deficiency of the mutant cells. Complementation of the Gal-deficient phenotype of Lec8 cells by Frc was occasionally found incomplete. This may reflect the localization of Frc to a limited subcompartment of the Golgi membrane, which was previously suggested to be the case in Drosophila.