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有明确的底物专一性。构建并分析了UDP-Gal转运体(UGT)和cMP-唾液酸转运体(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。我们以前的研究表明，UGT/CST嵌合体的第7个跨膜螺旋片段来自CST，它可以同时转运CMP-SIA和UDP-Gal。在本研究中，我们证明了CST第7个跨膜螺旋的N端一半对于嵌合转运体介导的CMPSIA的转运是必不可少的，并进一步成功地鉴定了位于该片段上的两个氨基酸残基，这两个氨基酸残基在识别CMPSIA作为转运底物方面特别重要。克隆并鉴定了与已知的UGTrel7转运体和果蝇FRC转运体密切相关的人UGTrel8转运体。利用异源表达系统检测UGTrel8在酿酒酵母中的底物特异性，确定其底物为UDP-GlcNAc。在CHO细胞中表达时，UGTrel8蛋白仅在高尔基体膜上表达，而UGTrel7蛋白定位于内质网。果蝇的多特异性UDP糖转运蛋白Frc转运蛋白是合成缺口低聚糖所必需的，而不是合成普通糖共轭化合物所必需的。将FRC基因导入UGT缺陷的Lec8细胞，观察FRC基因是否能弥补突变细胞的糖类缺陷。FRC对Lec8细胞半乳糖缺乏表型的补充有时是不完全的。这可能反映了FRC定位于高尔基体膜的一个有限的亚室，这一点以前被认为是在果蝇中的情况。

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.