Figure 1.

hNup133 localizes to the NPC and interacts with hNup107 in the yeast two-hybrid assay. (A) hNup133 localizes to the NPC. (a) Whole cell extracts from HeLa cells either nontransfected (1) or transfected with the pEGFP₃-hNup133 vector (2) were analyzed by Western blot using affinity purified anti-hNup133. Molecular mass markers are in kD. (b) Immunofluorescence analysis of methanol-fixed HeLa cells using affinity purified anti-hNup133 antibodies. Nuclear DNA was stained with DAPI. (c) Fluorescence analysis of living HeLa cells expressing the GFP₃-hNup133 fusion. The shape of the nucleus is revealed on the corresponding phase–contrast image. In b and c, cells were photographed in two different focal planes that reveal the discontinuous nuclear rim (left) and the punctuate staining on the surface of the nuclear envelope (middle). (B) S. cerevisiae, S. pombe, and human Nup133 interact with Nup84/Nup107 in a yeast two-hybrid assay. Strain CG194 was transformed with pASΔΔ plasmids expressing the Gal4p-BD either alone (control) or in fusion with scNup133, spNup133a, spNup133b, or hNup133 as indicated. Strain Y187 was transformed with pACTII plasmids expressing the Gal4p-AD either alone (C) or in fusion with the COOH-terminal domain of scNup84 (sc), spNup107 (sp), or hNup107 (h). Following mating, diploids expressing both plasmids were spotted onto minimum medium either lacking leucine and tryptophan (DO-LW) or lacking leucine, tryptophan, and histidine but containing 5 mM 3-aminotriazole (DO-LWH + 3AT), and the plates were incubated at 30°C for 3 d. Bars, 10 μm.