The N-terminal domain of Nab2 interacts with Mlp1. (a) A domain schematic of the Nab2 protein showing the Nab2 deletion mutants that were analyzed using the yeast two-hybrid assay. The relative locations of the N-terminal (N), QQQP repeat, RGG, and tandem zinc finger (CCCH) domains are indicated. The size (in amino acids) is indicated for the full-length protein, and the residues deleted from each variant are indicated. The yeast two-hybrid reporter strain (EGY48) was transformed with the indicated DBD plasmids expressing each Nab2 variant, in combination with an AD control plasmid (−), an AD plasmid expressing CT-Mlp1 (residues 1490–1875), or, as a control, an AD expressing Gfd1. Positive interactions are indicated by the blue color that arises from activation of the β-galactosidase (lacZ) reporter. (b) The N-terminal domain of Nab2 (Nab2-N) binds Mlp1 from yeast lysates. Purified recombinant GST-Nab2-N or GST alone as a control protein was incubated in yeast cell lysate prepared from either wild-type (WT) or MLP1 deletion (mlp1Δ) cells. The GST proteins were purified on glutathione beads, and copurifying proteins were visualized by Coomassie Blue staining. A band corresponding to Mlp1 copurified with GST-N from wild-type but not mlp1Δ cells. (c) GST-Nab2-N or GST alone as a control protein was incubated in yeast lysate from cells expressing TAP-tagged Mlp1. GST fusion proteins were purified, and unbound (U) and bound (B) fractions were analyzed by SDS-PAGE and immunoblotting with PAP antibody to detect TAP-tagged Mlp1. (d) Purified recombinant GST-Nab2-N or GST control protein was incubated with Mlp1 C-terminal domain and binding was assessed by Coomassie Brilliant Blue staining.