The Ndc1 interaction network for NPC assembly

用于 NPC 组装的 Ndc1 交互网络

• 批准号: 381447421
• 负责人: Professor Dr. Wolfram Antonin
• 金额: --
• 依托单位: Institut für Biochemie und Molekulare Zellbiologie
• 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/381447421?language=en
• 关键词: Ndc1; interaction; network; NPC; assembly

Nuclear pore complexes (NPCs) are the checkpoints of the nuclear envelope that mediate and control the selective passage between the interior of the cell nucleus and the cytoplasm. How these giant complexes assemble and integrate into the two membranes of the nuclear envelope remains a challenging research question. In humans, about 30 nucleoporins in numerous copies form these complexes consisting of about 1000 individual proteins, with many nucleoporins interacting with the pore membrane. In the last funding period, we identified and characterized an amphipathic helix in the C-terminal domain of Ndc1, an essential and highly conserved transmembrane protein of the NPC, as a membrane interaction motif. In S. cerevisiae, overexpression of this motif is toxic and dramatically alters intracellular membrane organization. This amphipathic helix functionally interacts with related motifs in the nucleoporins Nup53 and Nup59, which in turn are important for pore membrane binding and the connection of different NPC modules. Deletion of the amphipathic helix of Nup53 results in Ndc1 no longer being essential in yeast, suggesting a balanced interplay of amphipathic motifs in different nucleoporins in the assembly and/or function of NPCs. Since the amphipathic helix in Ndc1 is evolutionarily conserved, we will extend the work to vertebrates and, here, investigate the function of the amphipathic helix. We will test in Xenopus egg extracts and cells whether this motif is required for the function and assembly of NPCs at the end of mitosis and/or in interphase. In artificial membrane systems, we will characterize its membrane binding, e.g., preference for certain charged lipids of, for example, the pore membrane, and test whether it can bend membranes. We will solve the structure of this motif in the context of the C-terminal domain of Ndc1 to understand how the amphipathic helix interacts with and shapes the pore membrane. In the human NPC, about 250 amphipathic helices in six different nucleoporins (Nup160, Nup155, Nup153, Nup133, Nup53, and Ndc1) can interact with the pore membrane, and some of these motifs are important for different aspects of NPC assembly. These amphipathic helices have generally been studied biophysically in isolation, but their functional interplay has not been characterized. We will fill this knowledge gap by investigating the possible influences of the amphipathic motifs of Ndc1, Nup53, and Nup155, as all three proteins interact with each other, which is crucial for NPC assembly and function. For this purpose, we will use minimal membrane systems to dynamically analyze membrane curvatures and, in egg extracts, the possible mutual influence of the motifs on NPC assembly to understand their potential synergistic function in forming the saddle-like membrane shape of the NPC pore.

核孔复合物是核膜上的检查点，介导和控制细胞核内部与细胞质之间的选择性通道。这些巨大的复合物如何组装并整合到核膜的两层膜中仍然是一个具有挑战性的研究问题。在人类中，大约30个核孔蛋白以许多拷贝形成这些复合物，这些复合物由大约1000个单独的蛋白质组成，其中许多核孔蛋白与孔膜相互作用。在上一个资助期间，我们确定并表征了Ndc 1的C-末端结构域中的两亲性螺旋，Ndc 1是NPC的一种重要且高度保守的跨膜蛋白，作为膜相互作用基序。In S.在酿酒酵母中，该基序的过表达是有毒的，并显着改变细胞内膜组织。这种两亲性螺旋在功能上与核孔蛋白Nup 53和Nup 59中的相关基序相互作用，这反过来对孔膜结合和不同NPC模块的连接很重要。Nup 53的两亲性螺旋的缺失导致Ndc 1在酵母中不再是必需的，这表明在NPC的组装和/或功能中不同核孔蛋白中的两亲性基序的平衡相互作用。由于Ndc 1中的两亲性螺旋在进化上是保守的，我们将把这项工作扩展到脊椎动物，在这里，研究两亲性螺旋的功能。我们将在非洲爪蟾卵提取物和细胞中测试这个基序是否是有丝分裂结束时和/或间期NPC的功能和组装所必需的。在人工膜系统中，我们将表征其膜结合，例如，优选某些带电荷的脂质，例如孔膜，并测试其是否能使膜弯曲。我们将在Ndc 1的C-末端结构域的背景下解决这个基序的结构，以了解两亲性螺旋如何与孔膜相互作用并形成孔膜。在人NPC中，六种不同核孔蛋白（Nup 160，Nup 155，Nup 153，Nup 133，Nup 53和Ndc 1）中的约250个两亲性螺旋可以与孔膜相互作用，并且其中一些基序对于NPC组装的不同方面是重要的。这些两亲性螺旋通常被孤立地进行生物药理学研究，但它们的功能相互作用尚未被表征。我们将通过研究Ndc 1，Nup 53和Nup 155的两亲性基序的可能影响来填补这一知识空白，因为这三种蛋白质彼此相互作用，这对NPC的组装和功能至关重要。为了这个目的，我们将使用最小的膜系统来动态分析膜曲率，并在鸡蛋提取物中，NPC组装的基序可能的相互影响，以了解它们在形成鞍状膜形状的NPC孔中的潜在协同作用。