Nuclear import of histone H2A and H2B is mediated by a network of karyopherins - PubMed (original) (raw)

Nuclear import of histone H2A and H2B is mediated by a network of karyopherins

N Mosammaparast et al. J Cell Biol. 2001.

Abstract

The first step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.

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Figures

Figure 1

Figure 1

Kap114 binds to histones H2A and H2B in the cytoplasm. (a) Kap114-PrA and associated proteins were isolated from cytosol by IgG-Sepharose, eluted with a MgCl2 gradient, separated by SDS-PAGE, and visualized by Coomassie blue staining. The bands representing Kap114-PrA, TBP and histones H2A and H2B are indicated. (b) Cells expressing either H2A-PrA or H2B-PrA were fixed and the PrA moiety detected by indirect immunofluorescence. The coincident DAPI staining is shown. (c) H2A-PrA and associated proteins were isolated from cytosol. Bands representing Kap114p, Nap1p, H2A-PrA, and H2B are indicated. (d) H2B-PrA and associated proteins were isolated as described. Bands representing Kap114p, Nap1p, and H2B-PrA are indicated. Positions of molecular mass standards in kilodaltons are shown.

Figure 3

Figure 3

Nap1p is not required for the formation of a functional Kap114p-H2A/H2B complex. (a) Kap114-PrA was expressed in a Δ_nap1_ strain. Kap114-PrA and associated proteins were isolated as described and bands representing Kap114-PrA and H2A/H2B are indicated. Positions of molecular mass standards in kilodaltons are shown. (b) H2A-GFP2 or H2B-GFP2 were expressed in wild-type (WT) and Δ_nap1_ strains and detected by fluorescence imaging. The coincident Hoechst staining is shown.

Figure 2

Figure 2

Histone H2A and H2B both contain distinct NLSs. Different fragments of H2B (a) and H2A (b) (as indicated by amino acid number) were expressed as GFP2 fusions in wild-type cells, and the GFP moiety detected by fluorescence imaging. The coincident Hoechst staining is shown. Schematic of H2A and H2B drawn to scale; T, amino-terminal tail; α, alpha helix; αC, carboxy-terminal alpha-helix; CT, carboxy-terminal tail.

Figure 2

Figure 2

Histone H2A and H2B both contain distinct NLSs. Different fragments of H2B (a) and H2A (b) (as indicated by amino acid number) were expressed as GFP2 fusions in wild-type cells, and the GFP moiety detected by fluorescence imaging. The coincident Hoechst staining is shown. Schematic of H2A and H2B drawn to scale; T, amino-terminal tail; α, alpha helix; αC, carboxy-terminal alpha-helix; CT, carboxy-terminal tail.

Figure 4

Figure 4

H2A and H2B interact with additional Kaps in the absence of Kap114p. (a) H2A-PrA was expressed in a Δ_kap114_ strain. H2A-PrA and associated proteins were isolated as described. Bands representing potential Kaps (*), Nap1p, H2A-PrA, and H2B are indicated. Identical fractions were Western blotted with an antibody to Kap121p, shown below. (b) H2B-PrA was expressed in a Δ_kap114_ strain. H2B-PrA and associated proteins were isolated as described. Bands representing potential Kaps (*), Nap1p, and H2B-PrA are indicated. The fractions were Western blotted with antibodies to Kap121p and Kap123p as indicated below. (U) Unbound supernatant fraction. Positions of molecular mass standards in kilodaltons are shown.

Figure 5

Figure 5

Kap114p, Kap121p, Kap123p, and Kap95p participate in the nuclear import of H2A and H2B. H2A1-46GFP2 (a) or H2B1-52GFP2 (b) were expressed in wild-type (WT) and kap mutant strains (as indicated) and the GFP moiety detected by fluorescence imaging. The coincident Hoechst staining is shown. Strains were grown at 30°C, except for strains with temperature-sensitive alleles (kap121ts and kap95ts), which were grown at room temperature. Images were identically manipulated in Photoshop. (c) Images of the NLS reporter-bearing strains, grown as above or as indicated, were captured by fluorescence imaging. The mean fluorescence intensity of a defined pixel area was measured in the nucleus (N) and cytoplasm (C), and used to calculate the N:C ratio of mean fluorescence intensity. The mean ratio for 50 cells is shown (columns), as well as the SD.

Figure 5

Figure 5

Kap114p, Kap121p, Kap123p, and Kap95p participate in the nuclear import of H2A and H2B. H2A1-46GFP2 (a) or H2B1-52GFP2 (b) were expressed in wild-type (WT) and kap mutant strains (as indicated) and the GFP moiety detected by fluorescence imaging. The coincident Hoechst staining is shown. Strains were grown at 30°C, except for strains with temperature-sensitive alleles (kap121ts and kap95ts), which were grown at room temperature. Images were identically manipulated in Photoshop. (c) Images of the NLS reporter-bearing strains, grown as above or as indicated, were captured by fluorescence imaging. The mean fluorescence intensity of a defined pixel area was measured in the nucleus (N) and cytoplasm (C), and used to calculate the N:C ratio of mean fluorescence intensity. The mean ratio for 50 cells is shown (columns), as well as the SD.

Figure 5

Figure 5

Kap114p, Kap121p, Kap123p, and Kap95p participate in the nuclear import of H2A and H2B. H2A1-46GFP2 (a) or H2B1-52GFP2 (b) were expressed in wild-type (WT) and kap mutant strains (as indicated) and the GFP moiety detected by fluorescence imaging. The coincident Hoechst staining is shown. Strains were grown at 30°C, except for strains with temperature-sensitive alleles (kap121ts and kap95ts), which were grown at room temperature. Images were identically manipulated in Photoshop. (c) Images of the NLS reporter-bearing strains, grown as above or as indicated, were captured by fluorescence imaging. The mean fluorescence intensity of a defined pixel area was measured in the nucleus (N) and cytoplasm (C), and used to calculate the N:C ratio of mean fluorescence intensity. The mean ratio for 50 cells is shown (columns), as well as the SD.

Figure 6

Figure 6

Kap114p, Kap121p, and Kap95p bind directly to the H2A and H2B NLSs. (a) Reticulocyte lysate containing 35S-methionine–labeled Kap114p, Kap95p, Kap121p, or Kap108p was incubated with Sepharose-bound GST (GST), GST-H2A amino acids 1–46 (GST-H2A), or GST-H2B amino acids 1–52 (GST-H2B). The bound material was separated by SDS-PAGE. Kaps were visualized by fluorography of the gels (top four panels). The reticulocyte lysate input (2% of reaction) is also shown. The GST and GST-fusion proteins for a representative experiment, visualized by Coomassie blue staining, are shown (bottom). (b) 1 μg of purified, bacterially expressed Kap114p, Kap121p, or Kap95p was incubated with Sepharose-bound GST (GST), GST-H2A amino acids 1–46 (GST-H2A), or GST-H2B amino acids 1–52 (GST-H2B). Where indicated, the Kap was preincubated with 20 μM human RanGTP Q69L. The bound material was separated by SDS-PAGE and visualized by Coomassie blue staining. Positions of GST-fusion proteins are indicated; additional lower bands are probably degradation products.

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