Ramaen Odile, Joubert Alexandra, Simister Philip, Belgareh-Touzé Naïma, Olivares-Sanchez Maria Conception, Zeeh Jean-Christophe, Chantalat Sophie, Golinelli-Cohen Marie-Pierre, Jackson Catherine L, Biou Valérie, Cherfils Jacqueline
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Institut Jacques Monod-CNRS, Universités Paris VI et VII, 75251 Paris, France.
J Biol Chem. 2007 Sep 28;282(39):28834-28842. doi: 10.1074/jbc.M705525200. Epub 2007 Jul 19.
Guanine nucleotide exchange factors carrying a Sec7 domain (ArfGEFs) activate the small GTP-binding protein Arf, a major regulator of membrane remodeling and protein trafficking in eukaryotic cells. Only two of the seven subfamilies of ArfGEFs (GBF and BIG) are found in all eukaryotes. In addition to the Sec7 domain, which catalyzes GDP/GTP exchange on Arf, the GBF and BIG ArfGEFs have five common homology domains. Very little is known about the functions of these noncatalytic domains, but it is likely that they serve to integrate upstream signals that define the conditions of Arf activation. Here we describe interactions between two conserved domains upstream of the Sec7 domain (DCB and HUS) that determine the architecture of the N-terminal regions of the GBF and BIG ArfGEFs using a combination of biochemical, yeast two-hybrid, and cellular assays. Our data demonstrate a strong interaction between DCB domains within GBF1, BIG1, and BIG2 to maintain homodimers and an interaction between DCB and HUS domains within each homodimer. The DCB/HUS interaction is mediated by the HUS box, the most conserved motif in large ArfGEFs after the Sec7 domain. In support of the in vitro data, we show that both the DCB and the HUS domains are necessary for GBF1 dimerization in mammalian cells and that the DCB domain is essential for yeast viability. We propose that the dimeric DCB-HUS structural unit exists in all members of the GBF and BIG ArfGEF groups and in the related Mon2p family and probably serves an important regulatory role in Arf activation.
携带Sec7结构域的鸟嘌呤核苷酸交换因子（ArfGEFs）激活小GTP结合蛋白Arf，Arf是真核细胞中膜重塑和蛋白质运输的主要调节因子。在ArfGEFs的七个亚家族中，只有两个亚家族（GBF和BIG）存在于所有真核生物中。除了催化Arf上GDP/GTP交换的Sec7结构域外，GBF和BIG ArfGEFs还有五个共同的同源结构域。对于这些非催化结构域的功能知之甚少，但它们可能用于整合定义Arf激活条件的上游信号。在这里，我们结合生化、酵母双杂交和细胞分析，描述了Sec7结构域上游的两个保守结构域（DCB和HUS）之间的相互作用，这些相互作用决定了GBF和BIG ArfGEFs N端区域的结构。我们的数据表明，GBF1、BIG1和BIG2内的DCB结构域之间存在强烈相互作用以维持同二聚体，并且每个同二聚体内的DCB和HUS结构域之间存在相互作用。DCB/HUS相互作用由HUS框介导，HUS框是Sec7结构域之后大型ArfGEFs中最保守的基序。为支持体外数据，我们表明DCB和HUS结构域对于哺乳动物细胞中GBF1的二聚化都是必需的，并且DCB结构域对于酵母的生存能力至关重要。我们提出，二聚体DCB-HUS结构单元存在于GBF和BIG ArfGEF组的所有成员以及相关的Mon2p家族中，并且可能在Arf激活中起重要的调节作用。
