Nap1 and Chz1 have separate Htz1 nuclear import and assembly functions.

We analyzed the nuclear import and regulation of the yeast histone variant Htz1 (H2A.Z), and the role of histone chaperones Nap1 and Chz1 in this process. Copurification suggested that Htz1 and H2B dimerized in the cytoplasm prior to import. Like H2B, Htz1 contained a nuclear localization signal (NLS) in its N-terminus that is recognized by multiple karyopherins (also called importins), indicating multiple transport pathways into the nucleus. However, Kap114 and Kap123 appeared to play the major role in Htz1 import. We also identified a role for Nap1 in the import of Htz1/H2B heterodimers, and Nap1 formed a RanGTP-insensitive import complex with Htz1/H2B and Kap114. Nap1 was necessary for maintaining a soluble pool of Htz1, indicating that its chaperone function may be important for the dynamic exchange of histones within nucleosomes. In contrast, Chz1 was imported by a distinct import pathway, and Chz1 did not appear to interact with Htz1 in the cytoplasm. Genetic analysis indicated that NAP1 has a function in the absence of HTZ1 that is not shared with CHZ1. This provides further evidence that the histone chaperones Nap1 and Chz1 have separate Htz1-dependent and -independent functions.

Mutational analysis of H3 and H4 N termini reveals distinct roles in nuclear import.

Core histones H3 and H4 are rapidly imported into the nucleus by members of the karyopherin (Kap)/importin family. We showed that H3 and H4 interact with Kap123p, histone acetyltransferase-B complex (HAT-B), and Asf1p in cytosol. In vivo analysis indicated that Kap123p is required for H3-mediated import, whereas H4 utilizes multiple Kaps including Kap123p. The evolutionary conservation of H3 and H4 cytoplasmic acetylation led us to analyze the role of acetylation in nuclear transport. We determined that lysine 14 is critical for H3 NLS function in vivo and demonstrated that mutation of H3 lysine 14 to the acetylation-mimic glutamine decreased association with Kap123p in vitro. Several lysines in the H4 NLS are important for its function. We showed that mutation of key lysines to glutamine resulted in a greater import defect than mutation to arginine, suggesting that positive charge promotes NLS function. Lastly we determined that six of ten N-terminal acetylation sites in H3 and H4 can be mutated to arginine, indicating that deposition acetylation is not absolutely necessary in vivo. However, the growth defect of these mutants suggests that acetylation does play an important role in import. These findings suggest a model where cytosolic histones bind import karyopherins prior to acetylation. Other factors are recruited to this complex such as HAT-B and Asf1p; these factors in turn promote acetylation. Acetylation may be important for modulating the interaction with transport factors and may play a role in the release of histones from karyopherins in the nucleus.