Nup50/Npap60 function in nuclear protein import complex disassembly and importin recycling

Area of Science:

• Cell Biology
• Molecular Biology
• Structural Biology

Background:

• Nuclear import of proteins relies on the importin-alpha:beta complex binding cargo with nuclear localization signals (NLS).
• Importin-alpha:beta facilitates cargo passage through nuclear pores, with RanGTP mediating complex dissociation and importin recycling.
• Nucleoporin Nup50's role in vertebrate nuclear import has been proposed, potentially accompanying the import complex.

Purpose of the Study:

• To elucidate the precise function of nucleoporin Nup50 in the nuclear import pathway.
• To investigate the mechanism by which Nup50 interacts with importin-alpha and influences NLS binding.

Main Methods:

• Determined the crystal structure of the importin-alpha:Nup50 complex.
• Utilized mutagenesis to assess the functional importance of Nup50 binding sites on importin-alpha.
• Analyzed the structure of the Cse1p:Kap60p:RanGTP complex to infer Nup50 displacement.

Main Results:

• Nup50's N-terminal domain actively displaces NLSs from importin-alpha, suggesting a role in complex disassembly.
• Nup50 binds importin-alpha at two distinct sites: one overlapping the NLS-binding site and another at the C-terminus.
• Interaction at both sites is crucial for Nup50-mediated NLS displacement.

Conclusions:

• Nup50 actively participates in the terminal steps of nuclear protein import by promoting import complex disassembly.
• The findings provide a structural and mechanistic basis for understanding importin recycling and nuclear import regulation.