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

介导 GBF1 型植物 ARF-GEF 膜关联的域相互作用 â 机制和进化分析

• 批准号: 492659015
• 负责人: Professor Dr. Gerd Jürgens
• 金额: --
• 依托单位: Abteilung für Entwicklungsgenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/492659015?language=en
• 关键词: Domain; interactions; mediating; membrane; association

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.

蛋白质通过膜运输的亚细胞分布在真核生物的发育和生理过程中发挥着重要作用。转运囊泡的形成需要通过 ARF 鸟嘌呤核苷酸交换因子 (ARF-GEF) 催化的 GDP-GTP 交换激活小 ARF GTP 酶。大型 ARF-GEF，如哺乳动物中的 GBF1 或植物中的 GNOM，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 与 HDS3 连接；在这些结构域中，HUS 和 HDS1-3 结构域尚未得到功能表征。在哺乳动物 GBF1 中，DCB 结构域与相邻的 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 HUS、SEC7 和 HDS1 三个结构域）以实现 GNOM 的膜关联。结构域相互作用的差异与两个 ARF-GEF 之间的亚细胞作用位点的差异相关。 GBF1 作用于高尔基体，介导 COPI 囊泡的形成，以逆行转运至 ER，而 GNOM 在内体中发挥作用，是生长素外流载体 PIN1 极性再循环至基底质膜所必需的。本提案的目的是确定介导 GNOM 膜关联的域相互作用机制。我们将采用两种方法，这两种方法都涉及酵母相互作用实验和拟南芥功能研究。在进化策略中，将对来自不同真核物种的 ARF-GEF 进行分析，以确定一般 GBF1 型 ARF-GEF 和植物特异性 GNOM 型 ARF-GEF 之间的边界可能跨越真核生物。预期结果也具有实际意义。为了深入了解 GNOM 的结构域相互作用机制，我们将分析 GNOM 和最密切相关的通用 GBF1 型 ARF-GEF 贡献互补片段的嵌合蛋白。这样做是为了最大限度地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识别能够实现 GNOM 的 DCB-deltaDCB 相互作用的相关序列基序。计划中的实验可能会得到正在开发的 GNOM 结构模型的支持。除了这些定向改变之外，我们还提出了一种基于 GNOM 域随机突变的功能分析，用于识别交互面。此处，将选择一群诱变结构域来丧失 DCB-deltaDCB 相互作用；将检查突变结构域的全长，以确保它们只是失去了互补部分之间相互作用的能力。