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J. Biol. Chem., Vol. 280, Issue 18, 18442-18451, May 6, 2005

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Reconstitution of Nup157 and Nup145N into the Nup84 Complex^*

Malik Lutzmann, Ruth Kunze, Karin Stangl, Philipp Stelter, Katalin Fejes Tóth, Bettina Böttcher¶, and Ed Hurt||

From the Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany, the ^¶European Molecular Biology Laboratory, D-6900 Heidelberg, Germany, and the Kirchhoff-Institut für Physik, Universität Heidelberg, 69120 Heidelberg, Germany

About 30 different nucleoporins (Nups) constitute the nuclear pore complex. We have affinity-purified 28 of these nuclear pore proteins and identified new nucleoporin interactions by this analysis. We found that Nup157 and Nup170, two members of the large structural Nups, and the Gly-Leu-Phe-Gly nucleoporin Nup145N specifically co-purified with members of the Nup84 complex. In addition, Nup145N co-enriched during Nup157 purification. By in vitro reconstitution, we demonstrate that Nup157 and Nup145N form a nucleoporin subcomplex. Moreover, we show that Nup157 and Nup145N bind to the heptameric Nup84 complex. This assembly thus represents approximately one-third of all nucleoporins. To characterize Nup157 structurally, we purified and analyzed it by electron microscopy. Nup157 is a hollow sphere that resembles a clamp or a gripping hand. Thus, we could reconstitute an interaction between a large structural Nup, an FG repeat Nup, and a major structural module of the nuclear pore complex.

Received for publication, November 11, 2004 , and in revised form, January 28, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at www.jbc.org) contains supplemental Fig. 1 (growth analyses of yeast strains expressing TAP-tagged nucleoporins), Fig. 2 (peptide mass fingerprinting of newly identified protein interactions), and Fig. 3 (sedimentation equilibrium ultracentrifugation of Nup145N).

^|| Supported by Deutsche Forschungsgemeinschaft Grant SFB 638/B2 and a grant from Fonds der Chemischen Industrie and to whom correspondence should be addressed. Tel.: 49-6221-544173; Fax: 49-6221-544369; E-mail: cg5{at}ix.urz.uni-heidelberg.de.

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