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

Mosammaparast N, Jackson KR, Guo Y, Brame CJ, Shabanowitz J, Hunt DF, Pemberton LF - J. Cell Biol. (2001)

Bottom Line: Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Signaling, Department of Microbiology, Health Sciences Center, University of Virginia, 22908, USA.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169462&req=5

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.

Mentions: The observed mislocalization of H2A and H2B NLS reporters in various Kap mutant strains suggested that several Kaps may interact with these domains. The primary sequences of the H2A and H2B NLSs are not significantly similar, although they both contain several basic residues, characteristic of histone tails. We therefore used in vitro binding studies to determine whether Kap114p, as well as other Kaps, could interact with recombinant NLS domains from H2A and H2B. Approximately equal amounts of GST-H2A1-46, GST-H2B1-52, and GST alone were bound to glutathione-Sepharose. The Sepharose was incubated with reticulocyte lysate containing 35S-methionine-labeled Kap114p, Kap95p, Kap121p, or the control Kap, Kap108p. After washing and separating the bound proteins by SDS-PAGE, the gel was fluorographed. Kap114p bound to both the H2A and H2B NLSs, but not to GST alone (Fig. 6 a). Interestingly, Kap95p and Kap121p also bound to the H2A and H2B NLSs, but not to GST (Fig. 6 a), with more Kap95p binding to the H2A NLS than the H2B NLS. These results were consistent with the observed cytoplasmic mislocalization of the NLS-GFP fusions in the mutant strains (Fig. 5). Only low levels of binding to Kap108p could be detected, which is consistent with our biochemical and in vivo experiments, which do not implicate this Kap as a major import factor for either H2A or H2B.


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

Mosammaparast N, Jackson KR, Guo Y, Brame CJ, Shabanowitz J, Hunt DF, Pemberton LF - J. Cell Biol. (2001)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169462&req=5

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.
Mentions: The observed mislocalization of H2A and H2B NLS reporters in various Kap mutant strains suggested that several Kaps may interact with these domains. The primary sequences of the H2A and H2B NLSs are not significantly similar, although they both contain several basic residues, characteristic of histone tails. We therefore used in vitro binding studies to determine whether Kap114p, as well as other Kaps, could interact with recombinant NLS domains from H2A and H2B. Approximately equal amounts of GST-H2A1-46, GST-H2B1-52, and GST alone were bound to glutathione-Sepharose. The Sepharose was incubated with reticulocyte lysate containing 35S-methionine-labeled Kap114p, Kap95p, Kap121p, or the control Kap, Kap108p. After washing and separating the bound proteins by SDS-PAGE, the gel was fluorographed. Kap114p bound to both the H2A and H2B NLSs, but not to GST alone (Fig. 6 a). Interestingly, Kap95p and Kap121p also bound to the H2A and H2B NLSs, but not to GST (Fig. 6 a), with more Kap95p binding to the H2A NLS than the H2B NLS. These results were consistent with the observed cytoplasmic mislocalization of the NLS-GFP fusions in the mutant strains (Fig. 5). Only low levels of binding to Kap108p could be detected, which is consistent with our biochemical and in vivo experiments, which do not implicate this Kap as a major import factor for either H2A or H2B.

Bottom Line: Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Signaling, Department of Microbiology, Health Sciences Center, University of Virginia, 22908, USA.

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.

Show MeSH
Related in: MedlinePlus