Nuclear transport of histone 2b in mammalian cells is signal- and energy-dependent and different from the importin α/β-mediated process

Langer, Thomas

doi:10.1007/s004180000147

Cited by 19 publications

(13 citation statements)

References 63 publications

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“…Impβ is an exceptional transport receptor in that it not only binds transport substrates directly but also forms heterodimers with import adapters such as Impα or the import receptor Imp7. In contrast to published data (Langer, 2000), we demonstrate that H2B import can be mediated by the Impα/Impβ heterodimer in vitro. One explanation for this discrepancy is that Langer used nucleoplasmin core in the import buffer; in our hands, nucleoplasmin core blocks H2B import.…”

Section: Discussioncontrasting

confidence: 99%

Multiple pathways contribute to nuclear import of core histones

et al. 2001

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Nuclear import of the four core histones H2A, H2B, H3 and H4 is one of the main nuclear import activities during S-phase of the cell cycle. However, the molecular machinery facilitating nuclear import of core histones has not been elucidated. Here, we investigated the pathways by which histone import can occur. First, we show that core histone import can be competed by the BIB (β-like import receptor binding) domain of ribosomal protein L23a suggesting that histone import is an importin mediated process. Secondly, affinity chromatography on immobilized core histones revealed that several members of the importin β family of transport receptors are able to interact with core histones. Finally, we demonstrate that at least four known and one novel importin, importin 9, can mediate nuclear import of core histones into the nuclei of permeabilized cells. Our results suggest that multiple pathways of import exist to provide efficient nuclear uptake of these abundant, essential proteins.

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Section: Discussioncontrasting

confidence: 99%

Multiple pathways contribute to nuclear import of core histones

et al. 2001

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“…However, we have no evidence of Kapα binding to either the H2A or H2B NLS by PrA coimmunoprecipitation or by localization of the reporters in the Kapα ts strain, srp1-31 . This data correlates with experiments in mammalian cells, where H2B import was not blocked by addition of excess SV40 NLS peptide in a permeabilized cell system (Langer 2000), suggesting that histones are imported by an α-independent pathway.…”

Section: Discussionsupporting

confidence: 88%

Nuclear Import of Histone H2a and H2b Is Mediated by a Network of Karyopherins

Mosammaparast¹,

Jackson²,

Guo

et al. 2001

The Journal of Cell Biology

162

259

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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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“…Imp␤ family members are believed to play a critical role in nuclear import of histones, without the requirement for Imp␣ (14,15,24,25,(33)(34)(35). Here we utilized both new histone H2B proteins optimized for nuclear targeting, as well as those we have previously characterized (ref.…”

Section: Histone Octamers Reconstituted With Engineered H2b Proteins mentioning

confidence: 99%

“…1C, top left panel; Table 1). As each of the core histones contains an endogenous NLS that is recognized with high affinity by Imp␤ or its homologues (14,24,25,(33)(34)(35), there are potentially eight binding sites for Imp␤ in each octamer, reflected in the high maximal binding observed. Octamers containing H2B with GFP fused to its C terminus (H2B-GFP) also bound to Imp␤ with high affinity (K d ϭ3.1Ϯ0.7 nM; B max ϭ86,660Ϯ1059), demonstrating that the GFP tag can be effectively placed on either end of the histone, without affecting octamer formation or Imp binding (Fig.…”

Section: Histone Octamers Reconstituted With Engineered H2b Proteins mentioning

confidence: 99%

Efficient gene delivery using reconstituted chromatin enhanced for nuclear targeting

et al. 2008

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Nonviral gene delivery is hampered by difficulties associated with transporting negatively charged DNA through the cell membrane and, more importantly, the nuclear envelope of target cells. Here we show for the first time that chromatin reconstituted with histone H2B proteins optimized for nuclear targeting can be used as an efficient means to deliver DNA to the nucleus of intact living mammalian cells, resulting in high levels of transgene expression that were approximately 6-fold more than those achieved by commercial liposomal preparations. The high efficiency is due in part to DNA condensation and protection against degradation in the reconstituted chromatin, as well as its ability to interact with high affinity with the importin proteins of the cellular nuclear import machinery. "Chromofection," gene delivery by protein transduction using chromatin enhanced for nuclear targeting represents an efficient means to deliver DNA to a wide variety of cell types, with the potential to treat complex genetic disorders.

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Nuclear transport of histone 2b in mammalian cells is signal- and energy-dependent and different from the importin α/β-mediated process

Cited by 19 publications

References 63 publications

Multiple pathways contribute to nuclear import of core histones

Multiple pathways contribute to nuclear import of core histones

Nuclear Import of Histone H2a and H2b Is Mediated by a Network of Karyopherins

Efficient gene delivery using reconstituted chromatin enhanced for nuclear targeting

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