Dissecting the Interactions between NTF2, RanGDP, and the Nucleoporin XFXFG Repeats

doi:10.1074/jbc.275.8.5874

Dissecting the Interactions between NTF2, RanGDP, and the Nucleoporin XFXFG Repeats*

From the ^‡Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom and ^¶Max-Planck-Institut für Molekulare Physiologie, Otto Hahn Strasse 11, 44227 Dortmund, Germany

Abstract

We have used a range of complementary biochemical and biophysical methods to investigate the interactions between nuclear transport factor 2 (NTF2), the Ras family GTPase Ran, andXFXFG nucleoporin repeats that are crucial for nuclear trafficking. Microcalorimetry, microtiter plate binding, and fluorescence quenching in solution are all consistent with the binding constant for the NTF2-RanGDP interaction being in the 100 nm range, whereas the interaction between NTF2 andXFXFG repeat-containing nucleoporins such as Nsp1p is in the 1 μm range. Although the accumulation of NTF2 at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alter the affinity of NTF2 for RanGDP. These results indicate that, instead, Ran increases the binding of NTF2 to nucleoporins by another mechanism, most probably by Ran itself binding to nucleoporins and NTF2 binding to this nuclear pore-associated Ran.

Footnotes

↵* This work was supported by Research Grant RG0270/1998 from the Human Frontiers Science Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Recipient of EMBO Postdoctoral Fellowship ALTF98-121.
↵‖ Present address: Procter & Gamble European Service GmbH, Sulzbacher Str. 40, 65823 Schwalbach am Taunus, Germany.
↵** To whom correspondence should be addressed: Medical Research Council Laboratory of Molecular Biology, Hills Rd., Cambridge CB2 2QH, United Kingdom. Tel.: 44-1223-402463; Fax: 44-1223-213556; E-mail: ms@mrc-lmb.cam.ac.uk.
Abbreviations:

NTF2

nuclear transport factor 2

NPC

nuclear pore complex

NE

nuclear envelope

18R-NSP1

NSP1 protein construct containing 18XFXFG repeats

NBD-Cl

7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

NBD

4-nitrobenzo-2-oxa-1,3-diazole

PBS

phosphate-buffered saline
- Received November 18, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.