Nap1 ₂ •H2A-H2B•Kap114•Ran ^GTP effectively deposits H2A-H2B on tetrasomes. (A) Nucleosome assembly assay: H2A-H2B (H) pre-mixed with increasing concentrations of Nap1₂ (N₂) and/or Kap114 (K), without and with Ran^GTP (R), before the addition of ∼3–4 µM tetrasomes (TET; see Materials and methods). Samples were visualized using ethidium bromide- (top) and Coomassie-stained (middle) native PAGE gels. Nap1₂ allows nucleosome (NUC) formation while Kap114 inhibits it. Both Ran^GTP•Kap114•H2A-H2B and Nap1₂•H2A-H2B•Kap114•Ran^GTP form NUC effectively, but the former do not shield H2A-H2B from aggregation with DNA (white asterisks*). Bottom gel: control samples were without TET, where increasing the protein concentration did not affect mobility, indicating that protein complexes were stably formed at 1.5 µM. (B) Summary of protein concentrations and nucleosome assembly results. (C) Hypothetical model of how Nap1₂•H2A-H2B•Kap114•Ran^GTP promotes H2A-H2B transfer from Kap114 to assembling nucleosomes. The quaternary complex remains an effective chaperone of H2A-H2B and shields the bound histone from aberrant aggregation while the Kap114-bound Ran^GTP likely promotes histone release to the Kap114-bound Nap1₂, which can then effectively transfer H2A-H2B to tetrasomes to form nucleosomes. Source data are available for this figure: SourceData F6.