Shinotsuka Chisa, Yoshida Yusaku, Kawamoto Kazumasa, Takatsu Hiroyuki, Nakayama Kazuhisa
Institute of Biological Sciences and Gene Research Center, University of Tsukuba, 1-1-1 Tennohdai, Tsukuba Science City, Ibaraki 305-8572, Japan.
J Biol Chem. 2002 Mar 15;277(11):9468-73. doi: 10.1074/jbc.M112427200. Epub 2002 Jan 2.
BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.
BIG2是一种针对ADP核糖基化因子（ARF）家族小GTP酶的鸟嘌呤核苷酸交换因子（GEF），该家族小GTP酶调节COPI和衔接蛋白（AP）-1衣被蛋白复合物的膜结合。真菌代谢产物布雷菲德菌素A（BFA）可抑制ARF-GEF，并导致衣被蛋白从膜重新分布到细胞质，以及高尔基体复合物和反式高尔基体网络（TGN）的膜微管形成。为了研究BIG2的功能，我们检测了BIG2过表达对BFA诱导的ARF、衣被蛋白和细胞器标志物重新分布的影响。BIG2过表达阻断了BFA诱导的ARF1和AP-1复合物从膜上的重新分布，但未阻断COPI复合物的重新分布。这些观察结果表明，BIG2通过激活ARF参与AP-1的膜结合，但不参与COPI的膜结合。此外，不仅发现BIG2，而且发现ARF1和AP-1沿TGN发出的BFA诱导的膜微管呈球形肿胀队列。这些观察结果表明，BFA诱导的AP-1从TGN膜解离和TGN膜微管形成不是偶联事件，并提示细胞中存在ARF-GEF以外的BFA靶点。
