FIGURE 4.

Two basic segments within residues 5–20 and residues 29–34 of the histone H4 tail are used to bind Importins. A, alignment of the H3 IK-NLS motif with the H4 tail sequence. The H4 tail has an IK-NLS-like motif at residues 29–34. B, pulldown binding assays of immobilized GST-H4 tail proteins (wild type and the H4 tail(K5A/K8A/K12A/K16A/R17A/R19A/K20A) mutant) with Impβ, Kapβ2, Imp4, Imp5, Imp7, Imp9, and Impα (SDS-PAGE/Coomassie). C, pulldown binding assays of immobilized GST-H4 tail proteins (wild type and single site alanine mutants of Lys⁵, Lys⁸, Lys¹⁶, Arg¹⁷, Arg¹⁹, and Lys²⁰) with Importins (SDS-PAGE/Coomassie). Densities of the gel bands from experiments performed in triplicate are plotted in histograms. t tests were performed to assess effects of mutations (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001). Significance of mutations was also validated by one-way ANOVA tests (Impα, p > 0.05; Impβ, p ≤ 0.0001; Kapβ2, p ≤ 0.0001; Imp4, p ≤ 0.0001; Imp5, p > 0.05; Imp7, p ≤ 0.0001; Imp9, p ≤ 0.01). Error bars represent S.D. D, pulldown binding assays of immobilized GST-H4 tail proteins (wild type, H4 tail(I29A/T30A/K31A/P32A) mutant, and H4 tail(K5/K8/K12/K16/R17/R19/K20/²⁹ITKP³² to Ala) mutant) with Importins (SDS-PAGE/Coomassie).