Biochemical analysis of interactions between Kap114, Nap1, and H2A-H2B. (A) SEC-MALS analysis of Nap1 FL (N; blue), Kap114 (K; gray), and a 1:1 mixture of both (red). Left panel: The differential refractive index (dRI) traces are plotted as thin lines (left y-axis) and the molecular mass (kDa) traces as thick lines (right y-axis). The theoretical masses of the indicated proteins are marked with dashed lines. Right panel, Coomassie-stained SDS-PAGE of peak fractions. Results: As previously reported, N alone formed tetramers with the apparent molecular mass of ∼200 kDa (elution volume ∼10.3 ml). K alone eluted at ∼12.5 ml with the expected apparent molecular mass ∼110 kDa. A 1:1 molar mixture of K and N₂ formed a peak of ∼220 kDa that matches a K•N₂ complex. The increase in DRI signal of the K+N₂ compared to the N traces is consistent with incorporation of one K molecule. (B) SEC-MALS experiment for K (yellow) or N (cyan) binding to H2A-H2B (H) at the indicated molar ratios, plotted as in A. Results: K+H eluted with the expected molecular mass (elution volume ∼13 ml), whereas the N₂+H mixture eluted at volumes that span molecular masses of 150–200 kDa, possibly due to a mixture of N₂•H, N₂•H₂ and 2(N₂•H) complexes. (C) AUC titration and binding isotherm (inset) of Kap114 (K; 5.2 S) into the Nap1 core dimer (N₂) at the concentrations indicated. Molecular weight estimate, using the c(s) distribution of the most saturated 6:2 M ratio sample of the 7.9 S complex was 178 kDa, consistent with a K•N₂ complex (theoretical molecular weight, 186 kDa). The isotherm was generated using a one-site binding model and fitting residuals are plotted below. The dissociation constant or K_D is shown with the values in brackets representing a 95% confidence interval. (D) The full gel of one of the two binding assays shown in Fig. 1 B: 1 µM immobilized MBP-Kap114 and Nap1₂ core or FL ± 1 or 2 µM Sc H2A-H2B. Bound and unbound proteins after extensive washing were visualized by Coomassie-stained SDS-PAGE. Quantification of the average relative intensities of triplicate experiments of the bound FL Nap1, when normalized to the sample without H2A-H2B, is plotted with error bars that indicate standard deviation (SD). Unpaired, two-sided Student’s t test was performed. Data distribution was assumed to be normal but it was not formally tested. (E) Pull-down binding assay as in D, but with Xl H2A-H2B. Unlike Sc H2A-H2B, X. laevis (Xl) H2A-H2B increased Sc Nap1 association with Kap114, suggesting that different H2A-H2B homologs bind Sc Nap1 and Kap114 differently. Student t-test shows significant difference between 1 and 2 µM H2A-H2B samples, where less Nap1 was pulled down in the presence of excess H2A-H2B, suggesting destabilization of the ternary Kap114/Nap1₂/H2A-H2B complex. (F) Control pull-down experiment of 1 µM MBP (immobilized) and equimolar Nap1₂ ± H2A-H2B (1 or 2 M ratio). Background binding of Nap1 to the immobilized MBP was minimal. (G) SEC-MALS analysis of 1:1:1 (orange) or 1:1:2 (lilac) molar ratio K, N, and H mixtures, plotted as in A. Tabulated SEC-MALS results shown below. At 1:1:1 M ratio, most of the proteins assemble into a 1 Kap114/1 Nap1₂/1 H2A-H2B complex. There is a minor population of Kap114•H2A-H2B in peak 2, and thus there must be some excess Nap1₂ tetramers (∼200 kDa) in peak 1. When H2A-H2B is in excess; only Kap114•H2A-H2B, Nap1₂/H2A-H2B complexes formed, as the peak centers match the two traces in B. In summary, both pull-down assays and SEC-MALS analysis support that excess H2A-H2B destabilizes a 1:1:1 Kap114/Nap1₂/H2A-H2B ternary complex, dissociating it into binary Kap114•H2A-H2B and Nap12•H2A-H2B complexes. Ternary Kap114/Nap1₂/H2A-H2B interactions, such as in the cytoplasm, maybe most stable when all H2A-H2B heterodimers are adequately chaperoned. Source data are available for this figure: SourceData FS1.