Citterio Carmen, Vichi Alessandro, Pacheco-Rodriguez Gustavo, Aponte Angel M, Moss Joel, Vaughan Martha
Translational Medicine Branch and Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):2877-82. doi: 10.1073/pnas.0712224105. Epub 2008 Feb 14.
Guanine nucleotide-exchange factors (GEFs) activate ADP-ribosylation factor (ARF) GTPases that recruit coat proteins to membranes to initiate transport vesicle formation. Three mammalian GEFs are inhibited by brefeldin A (BFA). GBF1, predominantly associated with cis-Golgi membranes, functions early in the secretory pathway, whereas BIG1 and BIG2 act in trans-Golgi or later sites. Perturbation of endoplasmic reticulum (ER) functions can result in accumulation of unfolded or misfolded proteins that causes ER stress and unfolded protein response (UPR), with accumulation of ER stress response element (ERSE) gene products. BFA treatment of cells causes accumulation of proteins in the ER, ER stress, and ultimately apoptosis. To assess involvement of BFA-sensitive GEFs in the damage resulting from prolonged BFA treatment, HepG2 cells were selectively depleted of BIG1, BIG2, or GBF1 by using specific siRNA. Only GBF1 siRNA dramatically slowed cell growth, led to cell-cycle arrest in G(0)/G(1) phase, and caused dispersion of Golgi markers beta-COP and GM130, whereas ER structure appeared intact. GBF1 depletion also significantly increased levels of ER proteins calreticulin and protein disulfide isomerase (PDI). Proteomic analysis identified ER chaperones involved in the UPR that were significantly increased in amounts in GBF1-depleted cells. Upon ER stress, transcription factor ATF6 translocates from the ER to Golgi, where it is sequentially cleaved by site 1 and site 2 proteases, S1P and S2P, to a 50-kDa form that activates transcription of ERSE genes. Depletion of GBF1, but not BIG1 or BIG2, induced relocation of S2P from Golgi to ER with proteolysis of ATF6 followed by up-regulation of ER chaperones, mimicking a UPR response.
鸟嘌呤核苷酸交换因子（GEFs）激活ADP核糖基化因子（ARF）GTP酶，后者招募包被蛋白至膜上以启动运输小泡的形成。三种哺乳动物GEFs受布雷菲德菌素A（BFA）抑制。GBF1主要与顺式高尔基体膜相关，在分泌途径早期发挥作用，而BIG1和BIG2在反式高尔基体或更靠后的位点发挥作用。内质网（ER）功能的紊乱可导致未折叠或错误折叠蛋白的积累，从而引起ER应激和未折叠蛋白反应（UPR），同时ER应激反应元件（ERSE）基因产物也会积累。用BFA处理细胞会导致蛋白在ER中积累、ER应激，并最终引发细胞凋亡。为了评估BFA敏感的GEFs在长时间BFA处理所导致的损伤中的作用，通过使用特异性小干扰RNA（siRNA）选择性地敲低HepG2细胞中的BIG1、BIG2或GBF1。只有GBF1的siRNA显著减缓细胞生长，导致细胞周期停滞在G(0)/G(1)期，并引起高尔基体标志物β-COP和GM130的分散，而ER结构似乎保持完整。敲低GBF1还显著增加了ER蛋白钙网蛋白和蛋白二硫键异构酶（PDI）的水平。蛋白质组学分析鉴定出参与UPR的ER伴侣蛋白，其在敲低GBF1的细胞中的含量显著增加。在ER应激时，转录因子ATF6从ER转位至高尔基体，在那里它被位点1和位点2蛋白酶S1P和S2P依次切割成50 kDa的形式，从而激活ERSE基因的转录。敲低GBF1而非BIG1或BIG2会诱导S2P从高尔基体重新定位至ER，伴随ATF6的蛋白水解，随后ER伴侣蛋白上调，模拟了UPR反应。
