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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.

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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.
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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.

Mentions: It was possible that Kap114p interacted with either the NLS of H2A or H2B, whereas another Kap may recognize the other NLS. Hence, heterodimerization of H2A and H2B in the cytosol would result in the coisolation of both histones with Kap114-PrA. In addition, either histone would be able to coisolate all of the Kaps involved. To determine the direct role of Kap114p and the additional Kaps in the import of both histones in vivo, we analyzed the localization of the NLS-GFP2 constructs in strains bearing deletions or mutations in the KAP genes. The longer NLS constructs, H2A1-46GFP2 and H2B1-52GFP2, were used for these studies as they were the most nuclear in wild-type cells, but were unlikely to dimerize with endogenous histones. In addition, we quantitated the mean N:C ratios of GFP fluorescence intensity for both reporters in the different strains. In wild-type cells, these reporters were strictly nuclear with an N:C ratio of ∼3.5 (H2A) and ∼4.3 (H2B) (Fig. 5 c). In Δkap114 cells, both reporters were present in the nucleus, although there was more cytoplasmic signal than detected in wild-type cells. This correlated with the observed decrease in the N:C ratio compared with wild type (Fig. 5 c). Due to its coisolation with H2A and H2B, we were particularly interested in the role of Kap121p in histone import. In a kap121ts (pse1-1) strain, which has a temperature-sensitive allele of KAP121, significant mislocalization of both H2A and H2B NLS reporters was observed (Fig. 5, a and b), which correlated with a decrease in the N:C ratio (Fig. 5 c). This decrease was larger than that observed with Δkap114 and more pronounced with H2B. In addition, in a kap121ts/Δkap114 double-mutant strain, mislocalization of both reporters was even greater than in the single mutants, with much of the GFP signal localized to the cytoplasm (Fig. 5 a). This mislocalization was reflected in a further statistically significant decrease in the N:C ratio from that observed in the kap121ts strain. We also determined the effect of shifting these strains to 37°C for 1 h; however, the GFP localization appeared similar to unshifted cells (data not shown). These results show that both Kap121p and Kap114p function in the import of both H2A and H2B in vivo.


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, 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.
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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.
Mentions: It was possible that Kap114p interacted with either the NLS of H2A or H2B, whereas another Kap may recognize the other NLS. Hence, heterodimerization of H2A and H2B in the cytosol would result in the coisolation of both histones with Kap114-PrA. In addition, either histone would be able to coisolate all of the Kaps involved. To determine the direct role of Kap114p and the additional Kaps in the import of both histones in vivo, we analyzed the localization of the NLS-GFP2 constructs in strains bearing deletions or mutations in the KAP genes. The longer NLS constructs, H2A1-46GFP2 and H2B1-52GFP2, were used for these studies as they were the most nuclear in wild-type cells, but were unlikely to dimerize with endogenous histones. In addition, we quantitated the mean N:C ratios of GFP fluorescence intensity for both reporters in the different strains. In wild-type cells, these reporters were strictly nuclear with an N:C ratio of ∼3.5 (H2A) and ∼4.3 (H2B) (Fig. 5 c). In Δkap114 cells, both reporters were present in the nucleus, although there was more cytoplasmic signal than detected in wild-type cells. This correlated with the observed decrease in the N:C ratio compared with wild type (Fig. 5 c). Due to its coisolation with H2A and H2B, we were particularly interested in the role of Kap121p in histone import. In a kap121ts (pse1-1) strain, which has a temperature-sensitive allele of KAP121, significant mislocalization of both H2A and H2B NLS reporters was observed (Fig. 5, a and b), which correlated with a decrease in the N:C ratio (Fig. 5 c). This decrease was larger than that observed with Δkap114 and more pronounced with H2B. In addition, in a kap121ts/Δkap114 double-mutant strain, mislocalization of both reporters was even greater than in the single mutants, with much of the GFP signal localized to the cytoplasm (Fig. 5 a). This mislocalization was reflected in a further statistically significant decrease in the N:C ratio from that observed in the kap121ts strain. We also determined the effect of shifting these strains to 37°C for 1 h; however, the GFP localization appeared similar to unshifted cells (data not shown). These results show that both Kap121p and Kap114p function in the import of both H2A and H2B in vivo.

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