Mutational Analysis of H3 and H4 N Termini Reveals Distinct Roles in Nuclear Import

Abstract

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.

Recognition Elements in the Histone H3 and H4 Tails for Seven Different Importins

“…These results suggest that Lys 14 acetylation decreases H3 tail binding to Imp␤, Kap␤2, Imp4, Imp5, Imp7, Imp9, and Imp␣. In contrast, H4 tail diacetylation on Lys 5 and Lys 12 , marks of newly synthesized histones, has little effect on binding to most Importins.…”

“…A, pulldown binding assays of biotin-H3 tail (unacetylated) versus biotin-H3 tails acetylated at either Lys 14 or Lys 18 . B, pulldown binding assays of biotin-H4 tail (unacetylated) versus biotin-H4 tail acetylated at both Lys 5 and Lys 12 . Error bars represent S.D.…”

“…20 -30% of cytoplasmic H3 histones are acetylated at Lys 14 and/or Lys 18 , and all cytoplasmic H4 are acetylated at both Lys 5 and Lys 12 (30). However, the effect of H3 and H4 tail acetylation on histone import is controversial.…”

“…However, the effect of H3 and H4 tail acetylation on histone import is controversial. Mutations of H3 and H4 tail lysines to glutamines (acetylation mimics) in yeast abolished nuclear accumulation, slowed growth, or caused loss of viability, suggesting that acetylation impairs nuclear import (12). In contrast, H3 and H4 acetylation in P. polycephalum led to increased nuclear accumulation, and acetylated H4 tail peptides were reported to bind Imp4 better than unacetylated peptides, suggesting that acetylation may promote nuclear import (17,31).…”

“…Previous studies showed that N-terminal tails of histones H3 and H4 are necessary and sufficient for nuclear import, consistent with the presence of NLS-like sequences in the tails. Removal of either the H3 or H4 tail does not prevent nuclear import, but simultaneous removal of both tails produced non-viable S. cerevisiae and caused loss of nuclear H3-H4 in Physarum polycephalum (12,31). Of the Importins that bind and import H3 and H4, only classes of NLSs for Imp␣/Imp␤, Kap␤2, and Imp5 are known.…”