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J. Biol. Chem., Vol. 282, Issue 47, 33968-33976, November 23, 2007

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Molecular Determinants of Binding between Gly-Leu-Phe-Gly Nucleoporins and the Nuclear Pore Complex^*

Gary A. Ratner, Alec E. Hodel^¶, and Maureen A. Powers¹

From the Departments of Cell Biology and ^¶Biochemistry and the Biochemistry, Cell, and Developmental Biology Graduate Program, Emory University School of Medicine, Atlanta, Georgia 30322

The vertebrate nucleoporin Nup98 can be expressed in two distinct forms from differentially spliced mRNAs, either as a 98-kDa protein or as the 195-kDa Nup98/Nup96 polyprotein. Both forms undergo autoproteolytic processing to generate the 90-kDa Nup98 and either an 8-kDa tail or the nucleoporin Nup96. An equivalent cleavage event occurs in one yeast ortholog, Nup145, to produce Nup145N and Nup145C. We previously proposed that Nup145N, and possibly the other orthologs Nup116 and Nup100, might bind to Nup145C as demonstrated for Nup98 and Nup96. Here we have further investigated the interaction of both yeast and vertebrate Gly-Leu-Phe-Gly nucleoporins with the nuclear pore. We find that dynamic Nup98 binding can be recapitulated in vitro and that simultaneous translation and folding as a polyprotein are not required to allow subsequent binding between Nup98 and Nup96. We show that Nup145N and Nup145C do indeed bind to each other, and we have determined the dissociation constants for these interactions in vitro. Additionally, we characterize two sites of molecular interaction for each binding pair. Of the yeast orthologs, Nup116 binds far less robustly to Nup145C than does Nup145N, and Nup100 binding is barely detectable. Thus, we conclude that Nup116 and Nup100 likely use means of incorporation into the nuclear pore complex that are distinct from those used by Nup145N.

, and in revised form, September 20, 2007.

Note Added in Proof—Similarly, Lutzmann et al. found an interaction between Nup145N and Nup157 (Lutzmann, M., Kunze, R., Stangl, K., Stelter, P., Tóth, K. F., Böttcher, B., and Hurt, E. (2005) J. Biol. Chem. 280, 18442–18451). This heterodimer associated weakly with the highly stable Nup84 complex, most likely through the complex members Nup120 and Nup145C. However, they observed no interaction between Nup116 or Nup100 and either Nup157 or the Nup84 complex.

^* This work was supported by National Institutes of Health Grants GM58692 (to A. E. H. and M. A. P.) and GM59975 (to M. A. P.). 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.

Deceased.

¹ To whom correspondence should be addressed: Dept. of Cell Biology, Emory University, 615 Michael St., Atlanta, GA 30322. Tel.: 404-727-8859; Fax: 404-727-6256; E-mail: mpowers{at}cellbio.emory.edu.

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