Domain interactions mediating membrane association of GBF1-type plant ARF-GEFs – mechanistic and evolutionary analysis

蛋白质的亚细胞转运在真核生物的发育和生理过程中起着重要的作用。转运囊泡的形成需要通过由ARF鸟嘌呤-核苷酸交换因子（ARF- gef）催化的GDP-GTP交换激活小的ARF gtp酶。像哺乳动物中的GBF1或植物中的ggnm这样的大型arf - gef包含6个保守结构域：n端DCB结构域（二聚化）由HUS、SEC7 （GDP-GTP交换）和HDS1到HDS3连接；在这些结构域中，HUS和HDS1-3结构域尚未被功能表征。在哺乳动物GBF1中，DCB结构域与邻近的HUS结构域相互作用。相比之下，在gbf1相关的GNOM中，DCB结构域与互补的deltaDCB片段相互作用——需要三个结构域HUS、SEC7和HDS1——以实现GNOM的膜结合。结构域相互作用的差异与两种arf - gef之间亚细胞作用部位的差异有关。GBF1作用于高尔基体，介导COPI囊泡的形成，逆行运输至内质网，而GNOM作用于内体，是生长素外排载体PIN1向基质膜的极性再循环所必需的。本提案的目的是确定区域相互作用介导ggnom膜结合的机制。我们将采用酵母相互作用实验和拟南芥功能研究两种方法。在进化策略中，将分析来自不同真核生物物种的arf - gef，以确定一般gbf1型arf - gef和植物特异性gnom型arf - gef之间的边界可能在真核生物中运行。预期的结果也具有实际意义。为了深入了解ggnm结构域相互作用的机制，我们将分析ggnm和最密切相关的一般gbf1型ARF-GEF提供互补片段的嵌合蛋白。这样做是为了尽量减少由于巨大的进化距离而导致的潜在不兼容的风险。第二种方法的目的是确定GNOM的DCB-deltaDCB相互作用的相关序列基序。计划中的实验可能得到正在开发的GNOM结构模型的支持。除了这些定向改变外，我们还提出了一种基于GNOM结构域随机突变的功能分析，用于识别相互作用面。在这里，突变域的群体将被选择为DCB-deltaDCB相互作用的损失；将检查突变域的全长，以确保它们只是失去了在互补部分之间相互作用的能力。

The subcellular distribution of proteins by membrane traffic plays an important role in developmental and physiological processes of eukaryotes. The formation of transport vesicles requires activation of small ARF GTPases through GDP-GTP exchange which is catalysed by ARF guanine-nucleotide exchange factors (ARF-GEFs). Large ARF-GEFs like GBF1 in mammals or GNOM in plants comprise 6 conserved domains: The N-terminal DCB domain (dimerisation) is joined by HUS, SEC7 (GDP-GTP exchange) and HDS1 to HDS3; of these domains, HUS and HDS1-3 domains have not been functionally characterised. In mammalian GBF1, the DCB domain interacts with the adjacent HUS domain. By contrast, in GBF1-related GNOM, the DCB domain interacts with the complementary deltaDCB fragment – the three domains HUS, SEC7 and HDS1 are required – to enable membrane association of GNOM. The difference in domain interaction correlates with a difference in the subcellular site of action between the two ARF-GEFs. GBF1 acts at the Golgi apparatus, mediating the formation of COPI vesicles for the retrograde transport to the ER, whereas GNOM has a role at endosomes, being required for the polar recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane. The aim of this proposal is to identify the mechanism(s) of domain interaction mediating the membrane association of GNOM. We will pursue two approaches which both involve yeast interaction experiments and functional studies in Arabidopsis. In an evolutionary strategy, ARF-GEFs from different eukaryotic species will be analysed to determine where the boundary between general GBF1-type ARF-GEFs and plant-specific GNOM-type ARF-GEFs might run across the eukaryotes. The anticipated results also have practical implications. In order to gain insights into mechanisms of domain interaction of GNOM, we will analyse chimeric proteins to which GNOM and a most closely related general GBF1-type ARF-GEF contribute complementary segments. This will be done to minimise the risk of potential incompatibilities resulting from vast evolutionary distances. The aim of this second approach is to identify the relevant sequence motifs that enable the DCB-deltaDCB interaction of GNOM. The experiments planned might be supported by a structural model of GNOM that is being developed. In addition to these directed alterations, we propose a functional assay for the identification of interaction faces that is based on random mutagenesis of GNOM domains. Here, a population of mutagenised domains will be selected for the loss of DCB-deltaDCB interaction; the mutant domains will be checked for full length to make sure that they have merely lost their ability for interaction between the complementary parts.