Padilla Philip Ian, Pacheco-Rodriguez Gustavo, Moss Joel, Vaughan Martha
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2752-7. doi: 10.1073/pnas.0307345101. Epub 2004 Feb 18.
Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is an approximately 200-kDa brefeldin A-inhibited guanine nucleotide-exchange protein that preferentially activates ADP-ribosylation factor 1 (ARF1) and ARF3. BIG1 was found in cytosol in a multiprotein complex with a similar ARF-activating protein, BIG2, which is also an A kinase-anchoring protein. In HepG2 cells growing with serum, BIG1 was primarily cytosolic and Golgi-associated. After incubation overnight without serum, a large fraction of endogenous BIG1 was in the nuclei. By confocal immunofluorescence microscopy, BIG1 was localized with nucleoporin p62 at the nuclear envelope (probably during nucleocytoplasmic transport) and also in nucleoli, clearly visible against the less concentrated overall matrix staining. BIG1 was also identified by Western blot analyses in purified subnuclear fractions (e.g., nucleoli and nuclear matrix). Antibodies against BIG1, nucleoporin, or nucleolin coimmunoprecipitated the other two proteins from purified nuclei. In contrast, BIG2 was not associated with nuclear BIG1. Also of note, ARF was never detected among proteins precipitated from purified nuclei by anti-BIG1 antibodies, although microscopically the two proteins do appear sometimes to be colocalized in the nucleus. These data are consistent with independent intracellular movements and actions of BIG1 and BIG2, and they are also evidence of the participation of BIG1 in both Golgi and nuclear functions.
布雷菲德菌素A抑制的鸟嘌呤核苷酸交换蛋白1（BIG1）是一种分子量约为200 kDa的布雷菲德菌素A抑制的鸟嘌呤核苷酸交换蛋白，它优先激活ADP核糖基化因子1（ARF1）和ARF3。BIG1存在于细胞质中，与一种类似的ARF激活蛋白BIG2形成多蛋白复合物，BIG2也是一种A激酶锚定蛋白。在含血清生长的HepG2细胞中，BIG1主要位于细胞质和高尔基体相关区域。在无血清过夜培养后，大部分内源性BIG1位于细胞核中。通过共聚焦免疫荧光显微镜观察，BIG1与核孔蛋白p62定位于核膜（可能在核质运输过程中），也存在于核仁中，在整体染色较淡的背景下清晰可见。在纯化的亚核组分（如核仁与核基质）中，通过蛋白质免疫印迹分析也鉴定出了BIG1。抗BIG1、核孔蛋白或核仁素的抗体可从纯化的细胞核中共免疫沉淀出其他两种蛋白。相比之下，BIG2与细胞核中的BIG1不相关。同样值得注意的是，尽管在显微镜下有时这两种蛋白在细胞核中似乎共定位，但在用抗BIG1抗体从纯化的细胞核中沉淀的蛋白质中从未检测到ARF。这些数据与BIG1和BIG2在细胞内的独立移动和作用一致，也证明了BIG1参与高尔基体和细胞核功能。
