Substrate recognition and subcellular localization of nucleotide sugar transporters

核苷酸糖转运蛋白家族的每个成员都具有明确的底物特异性。构建并分析了UDP-Gal转运体（UGT）和cmp -唾液酸（CMP-Sia）转运体（CST）之间的分子嵌合体，以确定决定底物特异性的亚分子区域。我们之前的研究表明，从CST衍生出的第7跨膜螺旋片段的UGT/CST嵌合体可以同时转运CMP-Sia和UDP-Gal。在本研究中，我们发现CST第7跨膜螺旋的n端对嵌合转运体介导的CMP-Sia转运至关重要，并进一步成功鉴定了位于该段的两个氨基酸残基，这两个氨基酸残基在识别CMP-Sia作为转运底物时尤为重要。克隆并鉴定了人类UGTrel8转运蛋白，该转运蛋白与先前鉴定的UGTrel7转运蛋白和果蝇Frc转运蛋白密切相关。利用酿酒酵母的异源表达系统检测UGTrel8的底物特异性，确定其底物为UDP-GlcNAc。当在CHO细胞中表达时，UGTrel8蛋白仅在高尔基膜中表达，而UGTrel7蛋白则定位于内质网。Frc转运蛋白是一种来自果蝇的多特异性udp -糖转运蛋白，它是notch寡糖合成所必需的，但不用于一般糖缀合物的合成。将Frc cDNA转染到ugt缺陷的Lec8细胞中，观察Frc是否能弥补突变细胞的碳水化合物缺乏。偶尔发现Frc对Lec8细胞的gal缺陷表型的互补不完全。这可能反映了Frc定位于高尔基膜的一个有限的亚室，这在果蝇中被认为是这种情况。

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.