Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail-anchored proteins
Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail-anchored proteins
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DOI：
10.1111/tra.12550
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发表时间：
2018-03-01
期刊：
影响因子：
4.5
通讯作者：
Borgese, Nica
中科院分区：
文献类型：
--
作者：
Costa, Bruna Figueiredo;Cassella, Patrizia;Borgese, Nica
尾锚定（TA）蛋白通过不完全阐明的翻译后途径插入其靶细胞器。一些TA蛋白自发地插入到无蛋白质的脂质体中，但在体内靶向特定的细胞器。两种自发插入的细胞色素b5形式，b5-ER和b5-RR，其不同之处仅在于C-末端区域的电荷，分别靶向内质网（ER）或线粒体外膜（ERK）。为了弥合无细胞和细胞内结果之间的差距，我们分析了毛地黄皂苷透化的贴壁HeLa细胞中的靶向。在不存在胞质溶胶的情况下，b5-ER是两种b5形式的目的地，而在胞质溶胶中，b5-ER的C-末端负电荷决定靶向ER。跨膜识别复合物（TRC）途径的抑制仅部分降低了b5靶向，同时强烈影响经典的TRC底物小突触泡蛋白2（Syb 2）。为了确定其他途径，我们测试了一些小的抑制剂，发现Eeyarestatin I（ESI）减少了b5-ER和另一种自发插入TA蛋白的插入，而不影响Syb 2。该效应与已知的ESI、Sec 61和p97/VCP靶点无关。我们的研究结果表明，ER是自发插入TA蛋白的首选目的地，无论其C-末端电荷如何，并揭示了一种新的底物特异性ER靶向途径。
Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C-terminal negative charge of b5-ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI) reduced insertion of b5-ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI, Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and reveal a novel, substrate-specific ER-targeting pathway.