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Mol Biol Cell. 2008 May;19(5):2300-10. doi: 10.1091/mbc.E07-12-1279. Epub 2008 Feb 27.

The Nup358-RanGAP complex is required for efficient importin alpha/beta-dependent nuclear import.

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1
Department of Biochemistry I, Faculty of Medicine, Georg-August University of Göttingen, 37073, Göttingen, Germany.

Abstract

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.

PMID:
18305100
PMCID:
PMC2366868
DOI:
10.1091/mbc.E07-12-1279
[Indexed for MEDLINE]
Free PMC Article
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