Assembly and Preferential Localization of Nup116p on the Cytoplasmic Face of the Nuclear Pore Complex by Interaction with Nup82p

Abstract

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475–3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of C-terminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-Δ108 mutant after growth at 37°C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.

ACKNOWLEDGMENTS

We are indebted to numerous colleagues for generously sharing reagents: P. James for the two-hybrid strain and libraries; M. Bucci for the NUP82-GFP strain; C. Strambio-de-Castillia, G. Blobel, and M. Rout for the anti-Nup159p and anti-Pom152p monoclonal antibodies; M. Hurwitz and G. Blobel for the nup82-Δ108 strain. We also thank the members of the Wente lab for helpful discussions, and we thank L. Strawn and E. Ives for valuable comments on the manuscript.

This work was supported by grants to T.D.A. from the CRC (United Kingdom), to E.K. from the Wellcome Foundation, to K.J.R. from an NSRA, and to S.R.W. from the NIGMS.