Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p

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 resolv ed. 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 require ments distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belga reh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Bi ol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast ce ll lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Alth ough 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 specifica lly diminished in a nup82-Delta 108 mutant after growth at 37 degrees C. Im munoelectron microscopy analysis showed Nup116p was localized on both the c ytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest tha t Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasm ic Nup116p localization utilizing novel binding partners.