Anti-NUP50 monoclonal antibody, clone 358DU20.3.6

Nuclear import of proteins containing classical nuclear localization signals (NLS) is mediated by the importin-alpha:beta complex that binds cargo in the cytoplasm and facilitates its passage through nuclear pores, after which nuclear RanGTP dissociates the import complex and the importins are recycled. In vertebrates, import is stimulated by nucleoporin Nup50, which has been proposed to accompany the import complex through nuclear pores. However, we show here that the Nup50 N-terminal domain actively displaces NLSs from importin-alpha, which would be more consistent with Nup50 functioning to coordinate import complex disassembly and importin recycling. The crystal structure of the importin-alpha: Nup50 complex shows that Nup50 binds at two sites on importin-alpha. One site overlaps the secondary NLS-binding site, whereas the second extends along the importin-alpha C-terminus. Mutagenesis indicates that interaction at both sites is required for Nup50 to displace NLSs. The Cse1p: Kap60p: RanGTP complex structure suggests how Nup50 is then displaced on formation of the importin-alpha export complex. These results provide a rationale for understanding the series of interactions that orchestrate the terminal steps of nuclear protein import.

p27(Kip1) is a member of the Cip-Kip family of cyclin-dependent kinase (Cdk) inhibitors that binds to cyclin-Cdk complexes and inhibits their catalytic activity in response to antiproliferative stimuli. p27(Kip1) is regulated by several posttranscriptional mechanisms, including subcellular localization. We have identified a component of the nuclear pore complex (NPC), termed Nup50, through its two-hybrid interactions with p27(Kip1). Nup50 is a nucleoplasmically oriented component of the nuclear pore complex with a role in protein export (T. Guan, R. H. Kehlenbach, E. C. Schirmer, A. Kehlenbach, F. Fan, B. E. Clurman, N. Arnheim, and L. Gerace, Mol. Cell. Biol, 20:5619-5630, 2000). We found that murine Nup50 is a widely expressed nucleoporin and that Nup50 expression is highest in the developing neural tube and adult testes. We have also examined interactions between Nup50 and the NPC and found specific two-hybrid interactions between Nup50 and several well-defined components of the NPC, as well as coimmunoprecipitation of Nup50 with the nucleoporin Nup153 from transfected mammalian cells. In order to study Nup50 function in vivo, we cloned the mouse Nup50 genomic locus and created a targeted Nup50 deletion in the mouse germ line. Nup50 disruption resulted in a complex phenotype characterized by late embryonic lethality, neural tube defects, and intrauterine growth retardation. Although Nup50-null mouse embryo fibroblasts exhibited no defects in either cell cycle control or p27(Kip1) regulation, Nup50 deletion was associated with abnormalities in p27(Kip1) expression and cell proliferation in the developing neuroepithelium. We conclude that Nup50 is a nucleoporin with essential functions during mouse development.