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 (Nap1 (2) ) assemble into a 1:1:1 ternary complex. Cryogenic electron microscopy revealed two distinct Kap114/Nap1 (2) /H2A-H2B structures: one of H2A-H2B sandwiched between Nap1 (2) and Kap114, and another in which Nap1 (2) bound to the Kap114·H2A-H2B complex without contacting H2A-H2B. Another Nap1 (2) ·H2A-H2B·Kap114·Ran (GTP) 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 (2) together chaperone H2A-H2B better than either protein alone. When RanGTP is present, Kap114's chaperoning activity diminishes. However, the presence of Nap1 (2) within the Nap1 (2) ·H2A-H2B·Kap114·Ran (GTP) 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 (2) , ultimately directing its specific deposition into nucleosomes.