Analysis of ATF6 activation in Site-2 protease-deficient Chinese hamster ovary cells.
Analysis of ATF6 activation in Site-2 protease-deficient Chinese hamster ovary cells.
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DOI：
10.1247/csf.06015
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发表时间：
2006
影响因子：
1.5
通讯作者：
S. Nadanaka;H. Yoshida;R. Sato;K. Mori
中科院分区：
文献类型：
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作者：
S. Nadanaka;H. Yoshida;R. Sato;K. Mori
哺乳动物转录因子 ATF6 是组成型合成的，作为嵌入内质网 (ER) 的 II 型跨膜蛋白。当未折叠蛋白在内质网应激下通过称为调节膜内蛋白水解 (Rip) 的过程在 ER 中积累时，ATF6 被激活，在该过程中，ATF6 从 ER 转运至高尔基体，并在高尔基体中经历 Site-1 和 Site-2 蛋白酶的连续裂解。从高尔基体膜释放的 ATF6 胞质转录因子结构域进入细胞核，激活内质网定位的分子伴侣和折叠酶的转录，从而维持内质网的稳态。在这里，我们分析了 M19 细胞，这是一种缺乏 Site-2 蛋白酶的中国仓鼠卵巢细胞突变体。之前的研究表明，M19 细胞在诱导编码主要 ER 伴侣 BiP 的 mRNA 方面存在缺陷。在 M19 细胞中，ATF6 并未如预期那样从膜结合前体形式转化为裂解和核形式。此外，一些 ATF6 组成性地重新定位到高尔基体，在那里被 Site-1 蛋白酶切割，并保持与高尔基体的联系，表明 M19 细胞的 ER 受到组成性应激。与这一观点一致，另外两种 ER 应激反应介质 IRE1 和 PERK 在 M19 细胞中也被组成型激活。 M19 细胞显示模型蛋白的低效分泌。这些结果表明，Rip 介导的 ATF6 激活不仅对于 ER 应激细胞而且对于未应激细胞的 ER 稳态很重要。
Mammalian transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). It is activated when unfolded proteins are accumulated in the ER under ER stress through a process called regulated intramembrane proteolysis (Rip), in which ATF6 is transported from the ER to the Golgi apparatus where it undergoes sequential cleavage by Site-1 and Site-2 proteases. The cytosolic transcription factor domain of ATF6 liberated from the Golgi membrane enters the nucleus where it activates transcription of ER-localized molecular chaperones and folding enzymes, leading to the maintenance of the homeostasis of the ER. Here, we analyzed M19 cells, a mutant of Chinese hamster ovary cells deficient in Site-2 protease. It was previously shown that M19 cells are defective in the induction of mRNA encoding the major ER chaperone BiP. In M19 cells, ATF6 was not converted from the membrane-bound precursor form to the cleaved and nuclear form as expected. Moreover, some of the ATF6 was constitutively relocated to the Golgi apparatus, where it was cleaved by Site-1 protease, and remained associated with the Golgi apparatus, indicating that the ER of M19 cells was constitutively stressed. Consistent with this notion, the two other ER stress response mediators, IRE1 and PERK, were also constitutively activated in M19 cells. M19 cells showed inefficient secretion of a model protein. These results suggest that Rip-mediated activation of ATF6 is important for the homeostasis of the ER in not only ER-stressed but also unstressed cells.