Nap1 and Kap114 co-chaperone H2A-H2B and facilitate targeted histone release in the nucleus.

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 (Nap1 ) assemble into a 1:1:1 ternary complex. Cryogenic electron microscopy revealed two distinct Kap114/Nap1 /H2A-H2B structures: one of H2A-H2B sandwiched between Nap1 and Kap114, and another in which Nap1 bound to the Kap114·H2A-H2B complex without contacting H2A-H2B. Another Nap1 ·H2A-H2B·Kap114·Ran 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 Nap1 together chaperone H2A-H2B better than either protein alone. When RanGTP is present, Kap114's chaperoning activity diminishes. However, the presence of Nap1 within the Nap1 ·H2A-H2B·Kap114·Ran 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 Nap1 , ultimately directing its specific deposition into nucleosomes.