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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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Nap1p is not required for the formation of a functional Kap114p-H2A/H2B complex. (a) Kap114-PrA was expressed in a Δnap1 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 Δnap1 strains and detected by fluorescence imaging. The coincident Hoechst staining is shown.
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Figure 3: Nap1p is not required for the formation of a functional Kap114p-H2A/H2B complex. (a) Kap114-PrA was expressed in a Δnap1 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 Δnap1 strains and detected by fluorescence imaging. The coincident Hoechst staining is shown.

Mentions: These experiments suggested that both H2A and H2B may be import cargoes for the karyopherin, Kap114p. As these histones are imported as a heterodimer, we wished to determine whether one or both histones contained a functional NLS. Previous studies have predicted that H2B contains a basic NLS in the amino terminus, from residues 22–33 (Moreland et al. 1987). However, H2B lacking these amino acids was also imported, possibly via dimerization with H2A (Moreland et al. 1987). We expressed H2A and H2B fragments that did not include their dimerization domains to determine the precise location of the NLSs (the dimerization domain is located primarily in the alpha-2 helix, see schematic representations in Fig. 2, a and b; Luger et al. 1997). Full-length H2A and H2B were expressed as fusions with two GFP moieties (GFP2) and were strictly nuclear (shown in Fig. 3 b, below). In addition, GFP2 alone, with no NLS, appeared to be mainly cytoplasmic, with no detectable concentration in the nucleus (Fig. 2 a).


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)

Nap1p is not required for the formation of a functional Kap114p-H2A/H2B complex. (a) Kap114-PrA was expressed in a Δnap1 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 Δnap1 strains and detected by fluorescence imaging. The coincident Hoechst staining is shown.
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Related In: Results  -  Collection

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Figure 3: Nap1p is not required for the formation of a functional Kap114p-H2A/H2B complex. (a) Kap114-PrA was expressed in a Δnap1 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 Δnap1 strains and detected by fluorescence imaging. The coincident Hoechst staining is shown.
Mentions: These experiments suggested that both H2A and H2B may be import cargoes for the karyopherin, Kap114p. As these histones are imported as a heterodimer, we wished to determine whether one or both histones contained a functional NLS. Previous studies have predicted that H2B contains a basic NLS in the amino terminus, from residues 22–33 (Moreland et al. 1987). However, H2B lacking these amino acids was also imported, possibly via dimerization with H2A (Moreland et al. 1987). We expressed H2A and H2B fragments that did not include their dimerization domains to determine the precise location of the NLSs (the dimerization domain is located primarily in the alpha-2 helix, see schematic representations in Fig. 2, a and b; Luger et al. 1997). Full-length H2A and H2B were expressed as fusions with two GFP moieties (GFP2) and were strictly nuclear (shown in Fig. 3 b, below). In addition, GFP2 alone, with no NLS, appeared to be mainly cytoplasmic, with no detectable concentration in the nucleus (Fig. 2 a).

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