The Nup358-RanGAP complex is required for efficient importin alpha/beta-dependent nuclear import.
In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin alpha/beta-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin beta, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin beta strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore-associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin beta recycling and reformation of novel import complexes.
Hutten
Saskia
S
Department of Biochemistry I, Faculty of Medicine, Georg-August University of Göttingen, 37073, Göttingen, Germany.
Flotho
Annette
A
Melchior
Frauke
F
Kehlenbach
Ralph H
RH
D021581:Active Transport, Cell Nucleus; D000818:Animals; D000918:Antibody Specificity; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000378:metabolism; D022022:Nuclear Pore; D028861:Nuclear Pore Complex Proteins / Q000172:deficiency / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D028901:alpha Karyopherins / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D018832:Molecular Chaperones; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; C085844:Rangap1 protein, mouse; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C094493:ran-binding protein 2