Nup170C and Nup170N physically and functionally interact with each other. (A) Nup170C tethers Nup170N to the NE. The indicated GFP-tagged Nup170 domains were expressed under the control of the NOP1 promoter in the specified strains and analyzed by fluorescence microscopy. Cells are also shown by Nomarski optics. (B) Nup170C and Nup170N show a two-hybrid interaction. The indicated constructs, fused to either the GAL4 DNA–binding domain (G4BD) or the GAL4 activation domain (G4AD), were expressed into a reporter yeast strain. Cells were spotted in 10-fold serial dilution steps onto SDC-Trp-Leu (for plating efficiency) or SDC-Trp-Leu-Ade (for two-hybrid interaction) plates and incubated at 30°C for 3 d and 4 d, respectively. (C) Affinity-purified Nup170C coenriched Nup170N. Protein A (ProtA)–tagged Nup170C and protein A–tagged Nup82 were affinity purified from a yeast strain coexpressing myc-tagged Nup170N. The TEV-eluates were analyzed by SDS-PAGE and Coomassie staining (top) and Western blotting (bottom) using anti-myc antibodies to detect Nup170N. Coenriched Nsp1 and Nup159 are indicated on the right. The asterisks label the purified bait proteins, and the dot marks the TEV protease. Bar, 5 µm.