Skip to main content
. 2019 Mar 11;8:e43630. doi: 10.7554/eLife.43630

Figure 5. RanGTP modulates Imp9-H2A-H2B interactions for H2A-H2B deposition.

(A, B) DNA is titrated at 0.5, 1 and 2 molar equivalents of preformed Imp9•H2A-H2B (equimolar Imp9 and H2A-H2B mixed together) or RanGTP•Imp9•H2A-H2B (equimolar Imp9, H2A-H2B and RanGTP added together). Images of the same native gel, Coomassie stained in (A) and ethidium bromide stained in (B), are aligned for comparison. DNA cannot compete for H2A-H2B from the Imp9•H2A-H2B, leaving free DNA (B, increasing amounts from lanes 5 to 7) and intact Imp9•H2A-H2B (A, lanes 5–7). In contrast, DNA can compete for H2A-H2B from RanGTP•Imp9•H2A-H2B resulting in Imp9•RanGTP complexes (A, lanes 8–10), DNA•H2A-H2B complexes and very little free DNA (B, lanes 8–10). (C, D) Imp9 or Imp9•RanGTP (equimolar Imp9 and RanGTP added together) is titrated at 0.5–1.5 molar equivalents of H2A-H2B (in a DNA•H2A-H2B 1:7 complex). Images of the same native gel, ethidium bromide stained in (C) and Coomassie stained in (D), are aligned for comparison. Imp9 releases free DNA from DNA•H2A-H2B (C, lanes 3–6) and binds histones to form an Imp9•H2A-H2B complex (D, lanes 4–6). By comparison, Imp9•RanGTP releases little free DNA from DNA•H2A-H2B (C, lanes 7–10). (E) The presence of RanGTP and Imp9 facilitates H2A-H2B deposition onto the nucleosome. Nucleosome assembly assay where either H2A-H2B, Nap1•H2A-H2B, Imp9•H2A-H2B or RanGTP•Imp9•H2A-H2B is titrated in molar equivalents of 0.5 and 0.75 to tetrasome (TET; 2.5 µM). Nap1 and Imp9•RanGTP can form nucleosomes (NUC) while Imp9 cannot. Coomassie staining in Figure 5—figure supplement 1B. (F) Nucleosome disassembly assay where either Nap1, Imp9 or Imp9•Ran is titrated in molar equivalents of 0.5 and 0.75 to constant nucleosome (NUC; 2.5 µM). Imp9 can disassemble nucleosomes to tetrasomes while Nap1 and Imp9-Ran cannot. Coomassie staining in Figure 5—figure supplement 1C.

Figure 5.

Figure 5—figure supplement 1. RanGTP modulates Imp9-histones interaction for H2A-H2B deposition.

Figure 5—figure supplement 1.

(A) Controls for nucleosome assembly assay where either Nap1, Imp9 or Imp9•RanGTP is titrated in molar equivalents of 0.25, 0.5 and 0.75 to tetrasome (TET; 2.5 µM). These proteins do not alter the tetrasome. (B) Nucleosome assembly assay where either H2A-H2B, Nap1•H2A-H2B, Imp9•H2A-H2B or RanGTP•Imp9•H2A-H2B is titrated in molar equivalents of 0.5 and 0.75 to tetrasome (TET; 2.5 µM). Nap1 and Imp9•RanGTP can form nucleosomes (NUC) while Imp9 cannot. Gels are Coomassie stained (ethidium bromide stained gels are shown in Figure 5E). (C) Nucleosome disassembly assay where either Nap1, Imp9 or Imp9•RanGTP is titrated in molar equivalents of 0.5 and 0.75 to constant nucleosome (NUC; 2.5 µM). Imp9 can disassemble nucleosomes to tetrasomes while Nap1 and Imp9-RanGTP cannot. Gels are Coomassie stained (ethidium bromide stained gels are shown in Figure 5F).