Abstract

Core histones are synthesized and processed in the cytoplasm before transport into the nucleus for assembly into nucleosomes; however, they must also be chaperoned as free histones are toxic. The importin Kap114 binds and transports histone H2A-H2B into the yeast nucleus, where RanGTP facilitates H2A-H2B release. Kap114 and H2A-H2B also bind the Nap1 histone chaperone, which is found in both the cytoplasm and the nucleus, but how Nap1 and Kap114 cooperate in H2A-H2B processing and nucleosome assembly has been unclear. To understand these mechanisms, we used biochemical and structural analyses to reveal how Nap1, Kap114, H2A-H2B and RanGTP interact. We show that Kap114, H2A-H2B and a Nap1 dimer (Nap12) assemble into a 1:1:1 ternary complex. Cryogenic electron microscopy revealed two distinct Kap114/Nap12/H2A-H2B structures: one of H2A-H2B sandwiched between Nap12 and Kap114, and another in which Nap12 bound to the Kap114*H2A-H2B complex without contacting H2A-H2B. Another Nap12*H2A-H2B*Kap114*RanGTP structure reveals the nuclear complex. Mutagenesis revealed shared critical interfaces in all three structures. Consistent with structural findings, DNA competition experiments demonstrated that Kap114 and Nap12 together chaperone H2A-H2B better than either protein alone. When RanGTP is present, Kap114s chaperoning activity diminishes. However, the presence of Nap12 within the Nap12*H2A-H2B*Kap114*RanGTP quaternary complex restores its ability to chaperone H2A-H2B. This complex effectively deposits H2A-H2B into nucleosomes. Together, these findings suggest that Kap114 and Nap12 provide a sheltered path from cytoplasm to nucleus, facilitating the transfer of H2A-H2B from Kap114 to Nap12, ultimately directing its specific deposition into nucleosomes. Significance StatementFree histones are toxic and must be sequestered by other macromolecules in the cell. Nuclear import receptor Kap114 imports H2A-H2B into the nucleus while also chaperoning it. The histone chaperone Nap1 also chaperones H2A-H2B, but it is unclear how Nap1 and Kap114 cooperate to process H2A-H2B. We present biochemical and structural results that explain how Kap114, Nap1 and H2A-H2B assemble in the absence and presence of RanGTP, how Nap1 and Kap114 co-chaperone H2A-H2B, and how RanGTP and Nap1 coordinate the transfer of H2A-H2B from Kap114 to assembling nucleosomes in the nucleus.