The Ndc1 interaction network for NPC assembly

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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.