Fig. 6  A hydrophobic pocket on Nab2 is required for the interaction with Mlp1 but not with Gfd1. (a) Illustration of the hydrophobic putative protein interaction site (red) on the surface of the Nab2 N-terminal domain centered on Phe73 identified by the PPI-Pred site [http://www.bioinformatics.leeds.ac.uk/ppi-pred/]. The aromatic ring of Phe73 projects away from the protein surface and, hence, is an attractive candidate for a component of an interaction surface. (b) Wild-type (WT) or mutant (F72D, F73D, F73W, and F73A) GST-Nab2-N proteins were incubated with purified recombinant Mlp1 C-terminal domain. GST alone serves as a specificity control. GST fusion proteins were purified on glutathione beads and then eluted with sample buffer. Bound proteins were visualized by Coomassie Blue staining. (c) Wild-type (WT) or mutant (F72D and F73D) purified recombinant His-tagged Nab2-N was incubated with either Gfd1 beads (right) or control ovalbumin beads. Proteins bound to the beads were eluted with sample buffer, and the bound proteins were resolved by PAGE and visualized by Coomassie Blue staining. Note that Gfd1 and the control protein, ovalbumin, are covalently attached to beads; hence, they are not present in the bound fraction eluted with sample buffer. (d) Wild-type or mutant (F72D and F73D) GST-Nab2-N was incubated in yeast lysate prepared from cells expressing Mlp1-TAP. As a control, GST alone was also analyzed. Proteins that copurified with the GST fusion proteins on glutathione beads were analyzed by immunoblotting with PAP antibody, which detects the TAP-tagged Mlp1 protein.