Pull-down assays complemented the results obtained using the two-hybrid system and confirmed that the N-terminal domain of Nab2 without the QQQP domain is both necessary and sufficient to mediate interactions with Mlp1. A glutathione S-transferase (GST) fusion to the N-terminal domain of Nab2 (GST-Nab2-N, residues 1–97 of Nab2) was engineered, expressed in Escherichia coli, and purified. Either GST-Nab2-N or GST alone as a control protein was incubated in yeast lysate, and then, the GST protein and any associated proteins were bound to glutathione beads. As shown in Fig. 1b, a band of approximately 220 kDa that copurified with GST-Nab2-N but not with GST alone was visualized using Coomassie Blue staining. Immunoblotting confirmed that this band corresponded to the full-length Mlp1 protein (Fig. 1c). When the yeast lysate was prepared from mlp1Δ cells, this 220-kDa band was not detected by either Coomassie staining (Fig. 1b) or immunoblotting (Fig. 1c). Although the amount of Mlp1 copurified with GST-Nab2-N from yeast lysate was rather small, it is quite remarkable that a band corresponding to this full-length, very large, nuclear pore-associated protein17 can be isolated from yeast extract in a single step. The results of this experiment indicate that the N-terminal domain of Nab2 is sufficient to copurify Mlp1 from a complex mixture, showing that the interaction is highly specific.