Selective activation of the transcription factor ATF6 mediates endoplasmic reticulum proliferation triggered by a membrane protein
Selective activation of the transcription factor ATF6 mediates endoplasmic reticulum proliferation triggered by a membrane protein
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DOI：
10.1073/pnas.1101379108
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发表时间：
2011-05-10
影响因子：
11.1
通讯作者：
Borgese, Nica
中科院分区：
文献类型：
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作者：
Maiuolo, Jessica;Bulotta, Stefania;Borgese, Nica
众所周知，内质网（ER）能够响应货物负载和驻留膜蛋白表达的增加而扩大其表面积。尽管对增加的货物负载的反应（称为未折叠蛋白反应（UPR））已得到很好的表征，但对膜蛋白负载的反应机制尚不清楚。作为研究这一现象的模型系统，我们使用了 HeLa-TetOff 细胞系，该细胞系诱导表达尾部锚定结构，该结构由通过细胞色素 b5 [GFP-b(5)tail] 尾部锚定到内质网膜上的 N 端胞质 GFP 部分组成。去除多西环素后，GFP-b(5) 尾部以中等水平表达（总 ER 蛋白的 1-2%），然而，根据 EM 和磷脂酰胆碱合成增加三到四倍的评估，它会诱导 ER 增殖。我们研究了 UPR 三个臂中每一个臂的可能参与，发现在诱导 GFP-b(5)tail 表达后，只有激活转录因子 6 (ATF6) 臂被选择性激活； ATF6 α 激活峰值先于磷脂酰胆碱合成的增加。令人惊讶的是，在这些条件下没有观察到已知 ATF6 靶基因的上调。 ATF6 α 的沉默消除了 ER 增殖反应，而 Ire1 的敲低则没有效果。由于 GFP-b(5) 尾部缺少管腔结构域，因此我们观察到的响应不太可能源自 ER 管腔。相反，我们提出传感机制在脂质双层内运作以触发 ATF6 的选择性激活。
It is well known that the endoplasmic reticulum (ER) is capable of expanding its surface area in response both to cargo load and to increased expression of resident membrane proteins. Although the response to increased cargo load, known as the unfolded protein response (UPR), is well characterized, the mechanism of the response to membrane protein load has been unclear. As a model system to investigate this phenomenon, we have used a HeLa-TetOff cell line inducibly expressing a tail-anchored construct consisting of an N-terminal cytosolic GFP moiety anchored to the ER membrane by the tail of cytochrome b5 [GFP-b(5)tail]. After removal of doxycycline, GFP-b(5) tail is expressed at moderate levels (1-2% of total ER protein) that, nevertheless, induce ER proliferation, as assessed both by EM and by a three-to fourfold increase in phosphatidylcholine synthesis. We investigated possible participation of each of the three arms of the UPR and found that only the activating transcription factor 6 (ATF6) arm was selectively activated after induction of GFP-b(5)tail expression; peak ATF6 alpha activation preceded the increase in phosphatidylcholine synthesis. Surprisingly, up-regulation of known ATF6 target genes was not observed under these conditions. Silencing of ATF6 alpha abolished the ER proliferation-response, whereas knockdown of Ire1 was without effect. Because GFP-b(5) tail lacks a luminal domain, the response we observe is unlikely to originate from the ER lumen. Instead, we propose that a sensing mechanism operates within the lipid bilayer to trigger the selective activation of ATF6.