Makishima T, Nakashima T, Nagata-Kuno K, Fukushima K, Iida H, Sakaguchi M, Ikehara Y, Komiyama S, Nishimoto T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Higashi-ku, Fukuoka, Japan.
Genes Cells. 1997 Feb;2(2):129-41. doi: 10.1046/j.1365-2443.1997.1070303.x.
The tsBN7 cell line is one of the temperature-sensitive mutants for cell proliferation which have been isolated from the BHK21 cell line derived from the golden hamster. It has a mutation in the DAD1 gene encoding a 12.5kDa highly conserved protein through evolution, and enters apoptosis at the restrictive temperature due to this mutation.
DAD1 was recovered in light membrane fractions after differential centrifugation. It could not be released from the membrane, even by carbonate extraction, without a detergent. Upon digestion with proteinase K, both N and C terminal portions-but not the middle portions of DAD1- were released from the membrane. Thus, DAD1 appears to be an integral membrane protein in which both termini are located in the cytosol. DAD1 was localized in the endoplasmic reticulum. In accordance with a similarity to the yeast protein Ost2p, which is a subunit of the oligosaccharyltransferase, at the restrictive temperature, loss of DAD1 function caused a defect of N-linked glycosylation in tsBN7 cells resulting in apoptosis. However, tunicamycin, which is known to inhibit N-linked glycosylation did not induce apoptosis in either tsBN7 or BHK21 cells.
tsBN7 cells have a defect in N-linked glycosylation caused by the loss of DAD1.
tsBN7细胞系是从金黄仓鼠来源的BHK21细胞系中分离出的细胞增殖温度敏感突变体之一。它在编码一种进化上高度保守的12.5kDa蛋白质的DAD1基因中存在突变，并且由于这种突变在限制温度下进入凋亡状态。
通过差速离心，DAD1在轻膜组分中被回收。即使通过碳酸盐提取，如果没有去污剂，它也不能从膜上释放。用蛋白酶K消化后，DAD1的N端和C端部分——而不是中间部分——从膜上释放出来。因此，DAD1似乎是一种整合膜蛋白，其两端都位于胞质溶胶中。DAD1定位于内质网。与酵母蛋白Ost2p（寡糖基转移酶的一个亚基）相似，在限制温度下，DAD1功能的丧失导致tsBN7细胞中N-连接糖基化缺陷，从而导致凋亡。然而，已知抑制N-连接糖基化的衣霉素在tsBN7或BHK21细胞中均未诱导凋亡。
tsBN7细胞因DAD1缺失而存在N-连接糖基化缺陷。
