Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine Had-1 mutant cell lines and construction of deletion mutants
Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine Had-1 mutant cell lines and construction of deletion mutants
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DOI：
10.1093/oxfordjournals.jbchem.a022556
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发表时间：
1999-12-01
影响因子：
2.7
通讯作者：
Kawakita, M
中科院分区：
文献类型：
--
作者：
Ishida, N;Yoshioka, S;Kawakita, M
UDP-半乳糖转运蛋白是位于高尔基体的膜蛋白。它将UDP-半乳糖从胞质转运到高尔基体腔，从而为半乳糖基转移酶提供底物。我们通过分子克隆对鼠UDP-半乳糖转运蛋白进行了表征，目的如下：（i）阐明UDP-半乳糖转运活性缺陷的小鼠Had-1突变体的遗传缺陷的分子基础，和（ii）获得有助于我们计划合理方法以鉴定功能必需区域的信息，基于人和鼠UDP-半乳糖转运蛋白之间的一级结构的比较，我们鉴定了五个无义突变、一个错义Gly 178 Asp突变和两个异常剪接突变。虽然甘氨酸178在核苷酸-糖转运蛋白中高度保守，Gly 178 Ala变体是功能性的。人和鼠UDP-半乳糖转运蛋白之间的种属差异主要限于转运蛋白的N-和C-末端区域。在N-和C-末端区域的大量缺失不会导致UDP-半乳糖转运活性的丧失，表明这些胞质区域对于转运活性是不稳定的。该转运蛋白与绿色荧光蛋白在C-末端胞质尾部融合，而不损害任一蛋白的功能。我们的研究结果表明UDP-半乳糖转运蛋白的跨膜核心区的重要性。
UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters, We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations, Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.