Luminal Domain of ATF6 Alone Is Sufficient for Sensing Endoplasmic Reticulum Stress and Subsequent Transport to the Golgi Apparatus
Luminal Domain of ATF6 Alone Is Sufficient for Sensing Endoplasmic Reticulum Stress and Subsequent Transport to the Golgi Apparatus
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DOI：
10.1247/csf.10010
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发表时间：
2011-01-01
影响因子：
1.5
通讯作者：
Mori, Kazutoshi
中科院分区：
文献类型：
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作者：
Sato, Yoshimi;Nadanaka, Satomi;Mori, Kazutoshi
转录因子ATF6作为嵌入内质网（ER）的II型跨膜蛋白组成性合成。当未折叠的蛋白质在内质网中积累时，ATF6通过一种尚未确定的机制感知内质网应激，并重新定位到高尔基体，在那里通过Site-1和Site-2蛋白酶的连续作用将其切割，使释放的n端片段转运到细胞核中。这种由atf6介导的内质网定位分子伴侣和折叠酶的转录诱导，以及内质网相关降解成分，导致哺乳动物内质网稳态的维持。在这里，我们证明了ATF6的光腔结构域足以感知内质网应激并随后转运到高尔基体。ATF6的这个结构域被插入到n端信号序列和c端串联亲和纯化标签之间。由此产生的ATF6(C)-TAP转移到内质网，在那里它被糖基化和二硫键结合。ATF6(C)-TAP以单体和二聚体形式存在，半衰期相对较短，与全长ATF6相似。在二硫苏糖或thapsigarin诱导内质网应激时，内质网伴侣BiP与ATF6(C)-TAP分离，ATF6(C)-TAP转运到高尔基体，然后分泌到培养基中。Calnexin和蛋白二硫异构酶被鉴定为除BiP外还能与ATF6(C)-TAP结合的细胞蛋白，随后的分析显示蛋白二硫异构酶与ATF6(C)-TAP结合并具有伴侣活性。这些发现表明，ATF6(C)-TAP可以作为一种工具，分离出在内质网应激下护送ATF6从内质网到达高尔基体的蛋白。
The transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). When unfolded proteins accumulate in the ER, ATF6 senses such ER stress via an as yet undetermined mechanism and relocates to the Golgi apparatus where it is cleaved by sequential action of Site-1 and Site-2 proteases, allowing liberated N-terminal fragments to translocate into the nucleus. This ATF6-mediated transcriptional induction of ER-localized molecular chaperones and folding enzymes together with components of ER-associated degradation leads to the maintenance of ER homeostasis in mammals. Here, we demonstrated that the luminal domain of ATF6 alone is sufficient for sensing ER stress and subsequent transportation to the Golgi apparatus. This domain of ATF6 was inserted between the N-terminal signal sequence and C-terminal tandem affinity purification tag. The resulting ATF6(C)-TAP translocated into the ER, where it was glycosylated and disulfide bonded. ATF6(C)-TAP occurred as monomer and dimer, and exhibited a relatively short half-life, similar to full-length ATF6. On application of dithiothreitol-or thapsigargin-induced ER stress, the ER chaperone BiP dissociated from ATF6(C)-TAP, and ATF6(C)-TAP was transported to the Golgi apparatus and then secreted into medium. Calnexin and protein disulfide isomerase were identified as cellular proteins capable of binding to ATF6(C)-TAP in addition to BiP, and subsequent analysis revealed that protein disulfide isomerase was bound to ATF6(C)-TAP with chaperone activity. These findings indicate that ATF6(C)-TAP can be used as a tool to isolate protein(s) that escort ATF6 from the ER to the Golgi apparatus in response to ER stress.