Figure 3.

Htz1, Nap1, Kap114 form a co-complex. (A) The Htz1 NLS (residues 1–53) or Asf1 GFP₂ reporter was expressed in yeast strains containing mutations of individual KAP genes as indicated and localized by fluorescent microscopy. Coincident Hoechst staining is shown. (B) Recombinant GST-Htz1_1–53 (250 nM) was immobilized on glutathione sepharose beads and incubated with the indicated recombinant Kap protein (MBP-Kap114 100 nM, MBP-Kap123 500 nM, Kap95 500 nM). MBP-LacZ (500 nM) was utilized as a negative control. After extensive washing, bound protein was analyzed by SDS-PAGE and Coomassie blue staining (CBB). A 50% input of MBP-LacZ is shown. * indicates a proteolytic product of MBP-Kap123. (C) Recombinant GST-Htz1_1–53 (400 nM) was immobilized on glutathione sepharose beads and incubated with either recombinant Nap1 (250 nM) or H6-PrA-Chz1 (500 nM). Similarly, GST (500 nM) was incubated with Nap1 (250 nM) as a control. After washing, protein was eluted from the beads and analyzed by SDS-PAGE and Coomassie stain. 50% input of Nap1 and H6-PrA-Chz1 are indicated. (D) Recombinant GST-Htz1_1–53 (400 nM) was immobilized on glutathione sepharose in the presence or absence of Nap1 (250 nM). MBP-Kap114 (100 nM) or MBP-Kap123 (300 nM) was pre-incubated with or without H6-Gsp1Q71L-GTP (50 μM) and/or MBP-LacZ (400nM) and then added to the binding reaction. After extensive washing, bound protein was analyzed by SDS-PAGE and Coomassie stain.