Shen Xiaoyan, Xu Kai-Feng, Fan Qingyuan, 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. 2006 Feb 21;103(8):2635-40. doi: 10.1073/pnas.0510599103. Epub 2006 Feb 13.
ADP-ribosylation factors (ARFs) are critical in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)1 and BIG2 activate ARFs by accelerating replacement of bound GDP with GTP. Additional and differing functions of these approximately 200-kDa proteins are now being recognized, as are their independent intracellular movements. Here, we describe the localization in COS7 cells by immunofluorescence microscopy of BIG2, but not BIG1, with structures that have characteristics of recycling endosomes during transferrin (Tfn) uptake and Tfn receptor (TfnR) recycling. Cell content of BIG2 and Rab11, but not TfnR, BIG1, Rab4, or Exo70, was increased after 60 min of Tfn uptake. BIG2, but not BIG1, appeared in density-gradient fractions containing TfnR, Rab11, and Exo70 after 60 min of Tfn uptake. Treatment of cells with BIG2 small interfering RNA (siRNA), but not BIG1 or control siRNAs, decreased BIG2 protein >90% without affecting BIG1, ARF, or actin content, whereas TfnR was significantly increased as was its accumulation in perinuclear recycling endosomes. Tfn release appeared unaffected by BIG1 siRNA but was significantly slowed from cells treated with BIG2 siRNA alone or plus BIG1 siRNA. We suggest that BIG2 has an important role in Tfn uptake and TfnR recycling, perhaps through its demonstrated interaction with Exo70 and the exocyst complex.
ADP核糖基化因子（ARFs）在囊泡运输中起关键作用。布雷菲德菌素A抑制的鸟嘌呤核苷酸交换蛋白（BIG）1和BIG2通过加速将结合的GDP替换为GTP来激活ARFs。现在人们认识到这些大约200 kDa的蛋白质还有其他不同的功能，以及它们独立的细胞内运动。在这里，我们通过免疫荧光显微镜描述了BIG2而非BIG1在COS7细胞中的定位，其与转铁蛋白（Tfn）摄取和转铁蛋白受体（TfnR）循环过程中具有再循环内体特征的结构相关。摄取Tfn 60分钟后，BIG2和Rab11的细胞含量增加，但TfnR、BIG1、Rab4或Exo70的细胞含量未增加。摄取Tfn 60分钟后，BIG2而非BIG1出现在含有TfnR、Rab11和Exo70的密度梯度组分中。用BIG2小干扰RNA（siRNA）而非BIG1或对照siRNAs处理细胞，可使BIG2蛋白减少>90%，而不影响BIG1、ARF或肌动蛋白含量，而TfnR及其在核周再循环内体中的积累显著增加。Tfn的释放似乎不受BIG1 siRNA的影响，但单独用BIG2 siRNA或加BIG1 siRNA处理的细胞中Tfn的释放明显减慢。我们认为BIG2在Tfn摄取和TfnR循环中起重要作用，可能是通过其与Exo70和外泌体复合物的相互作用来实现的。
