Nup50 function in nuclear trafficking

• Applicant: Dr. Guillaume Holzer
• Subject Area: Cell Biology ; Biochemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 507747856

Nuclear pore complexes (NPCs) control the passage of macromolecules between the nucleus and the cytoplasm. NPCs are composed of about 30 distinct proteins called nucleoporins (Nups), found in many copies per NPC, making them one of the largest protein complex of the cell. Nuclear trafficking is paramount for cellular homeostasis, and NPC malfunctions are implicated in a wide spectrum of diseases such as cancer or neurological disorders.In a recent publication, I have shown that Nup50 is required for NPC assembly. Surprisingly, while Nup50 NPC localization is not required for its function in NPC assembly, its newly described interaction with and stimulation of RCC1, a guanine nucleotide exchange factor (GEF) for Ran, is critical.RCC1 GEF activity is crucial for nuclear trafficking and Nup50 interacts with proteins involved in nuclear import (Importins, Ran). In this project, I will evaluate the role of Nup50 in nuclear trafficking addressing three questions: I) Does the Nup50 mediated increase in RCC1 GEF activity contribute to nuclear trafficking? II) Does the binding of Ran to Nup50 support nuclear trafficking? III) Does Nup50 localization at the nuclear side of NPCs enhance nuclear trafficking efficiency? For this, I will first characterize the Nup50/RCC1/Importin α/RanGTP interaction network. Can these proteins form a tetrameric complex? If not, which associations are possible, which are mutually exclusive? Secondly, I will determine, in collaboration with Prof. Thomas Schwartz (MIT, Cambridge, USA), the structure of at least the Nup50/RCC1 dimer and ideally the tetrameric complex. Next, I will evaluate the role of the Nup50 interaction network in nuclear protein import. I will use Xenopus egg extracts, where nuclei can be assembled in vitro, profiting from the high flexibility of this cell free system. NPCs formed with Nup50 versions defective in its distinct functions (RCC1, Importin α or Ran binding, NPC localization) will be assayed for their nuclear import capabilities. Findings in Xenopus extract assay will be validated in cells, by replacing endogenous Nup50 with relevant Nup50 mutant versions and quantitatively analyze nuclear import efficiency. Lastly, I will compare the nuclear and cytoplasmic protein profile of wild type and Nup50 knockdown cells to investigate which protein localizations are most severely impacted by Nup50 malfunction. Together, this project will dissect the role of Nup50 and its interacting partner in nuclear trafficking, fundamental for a better understanding this function in cellular homeostasis and pathologies. Moreover, this project might spark hypothesis regarding Nup50 roles in other cellular functions such as cell cycle control or DNA repair, which are currently poorly understood.

• DFG Programme: Research Grants