title: Ran-dependent docking of importin-beta to RanBP2/Nup358 filaments is essential for protein import and cell viability.
abstract: RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)-mediated protein import, nuclear export signal-mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)-importin-β, accumulation of importin-β at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP-importin-β complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.
authors.affiliation: Department of Biochemistry and Molecular Biology and 2 Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hamada
authors.forename: Anna
authors.initials: A
authors.lastname: Haeger
authors.forename: Karthik B
authors.initials: KB
authors.lastname: Jeganathan
authors.forename: Janine H
authors.initials: JH
authors.lastname: van Ree
authors.forename: Liviu
authors.initials: L
authors.lastname: Malureanu
authors.forename: Sarah
authors.initials: S
authors.lastname: Wälde
authors.forename: Jomon
authors.initials: J
authors.lastname: Joseph
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Kehlenbach
authors.forename: Jan M
authors.initials: JM
authors.lastname: van Deursen
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D020090:Chromosome Segregation; D003599:Cytoskeleton / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008938:Mitosis; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019913:Nuclear Localization Signals / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C494045:M9 nuclear localization signal peptide; D018832:Molecular Chaperones; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D012333:RNA, Messenger; C102460:Ran protein, mouse; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D028961:beta Karyopherins; C094493:ran-binding protein 2; D044767:Ubiquitin-Protein Ligases; D020931:ran GTP-Binding Protein