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MBC in Press, published online ahead of print February 27, 2008
Mol. Biol. Cell 10.1091/mbc.E07-12-1279

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Submitted on December 26, 2007
Revised on February 1, 2008
Accepted on February 15, 2008

The Nup358-RanGAP Complex is Required for Efficient Importin {alpha}/{beta}-dependent Nuclear Import

Saskia Hutten, Annette Flotho, Frauke Melchior, and Ralph H. Kehlenbach

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

Monitoring Editor: Karsten Weis

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. While 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.

Address correspondence to: Ralph H. Kehlenbach (rkehlen{at}gwdg.de)






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