Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

Lu, Quanlong; Lu, Zhigang; Liu, Qinying; Guo, Li; Ren, He; Fu, Jingyan; Jiang, Qing; Clarke, Paul R.; Zhang, Chuanmao

doi:10.1038/cr.2012.113

Cited by 16 publications

(13 citation statements)

References 45 publications

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“…When recombinant TopBP1 was added back to TopBP1-depleted extracts before nuclear envelope assembly, a small amount of TopBP1 may participate nuclear envelope assembly and is sufficient for its role in DNA replication, consistent with previous findings [26,60,61]. A portion of TopBP1 in the vicinity of demembranated chromatin may bind to chromatin and participate nuclear envelope assembly in a similar way as other NLS-containing proteins such as nucleoplasmin and histones [62]. However, compromised nuclear import of TopBP1 leads to deficiency in checkpoint activation (Fig.…”

Section: Discussionsupporting

confidence: 87%

Importin β-dependent nuclear import of TopBP1 in ATR–Chk1 checkpoint in Xenopus egg extracts

Bai¹,

Michael²,

Shan³

2014

Cellular Signalling

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TopBP1, a multiple-BRCT-containing protein, plays diverse functions in DNA metabolism including DNA replication, DNA damage response and transcriptional regulation. The cytoplasmic localization of TopBP1 has been found associated with breast cancer susceptibility in clinical studies, suggesting the biological significance of TopBP1’s sub-cellular localization. However, it remains elusive how TopBP1 is shuttled into nucleus and recruited to chromatin under normal or stressful conditions. Taking advantage of Xenopus egg extract, we identified Importin β as a new interacting protein of the TopBP1 C-terminus. We verified the TopBP1-Importin β association via GST pulldown and coimmunoprecipitation assays. We then demonstrated that TopBP1’s C-terminal motif (designated as CTM, 23 amino acids) containing a putative NLS (Nuclear Localization Signal) was required for Importin β interaction and that CT100 of Importin β (100 amino acids of extreme C-terminus of Importin β) was required for TopBP1 interaction. Further structure-function analysis reveals that the CTM of TopBP1 is essential for TopBP1’s nuclear import and subsequent chromatin recruitment, thereby playing important roles in DNA replication and mitomycin C (MMC)-induced Chk1 phosphorylation. In addition, Importin β-specific inhibitor importazole inhibits TopBP1’s nuclear import and the MMC-induced Chk1 phosphorylation. With ongoing DNA replication, the Importin β-dependent nuclear import of TopBP1 was indeed required for the MMC-induced Chk1 phosphorylation. Our data also suggest that checkpoint activation requires more TopBP1 than DNA replication does. The requirement of TopBP1’s CTM motif for ATR-Chk1 checkpoint can be bypassed in a nucleus-free AT70 system. Taken together, our findings suggest the CTM motif-mediated TopBP1 shuttling into nucleus via Importin β plays an important role in the ATR-Chk1 checkpoint signaling in Xenopus egg extracts.

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Section: Discussionsupporting

confidence: 87%

Importin β-dependent nuclear import of TopBP1 in ATR–Chk1 checkpoint in Xenopus egg extracts

Bai¹,

Michael²,

Shan³

2014

Cellular Signalling

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“…Electron microscopy processing in RPE cells and zebrafish embryos were carried out as previously described 54 , 56 . Briefly, cells or zebrafish embryos were fixed in a solution of 2% glutaraldehyde with or without 4% paraformaldehyde in 0.1 M sodium cacodylate buffer followed by post-fixation with 1% Osmium tetroxide and 1% Uranyl acetate.…”

Section: Methodsmentioning

confidence: 99%

Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation

et al. 2015

Self Cite

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Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11-Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to pre-ciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle (CV) formation from smaller distal appendage vesicles (DAV). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated CV assembly. Interestingly, only after CV assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step whereby EHD1 and EHD3 reorganize the M-centriole and associated DAV prior to coordinated ciliary membrane and axoneme growth.

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“…To test the possibility that eNpm may have an altered threshold concentration for histone deposition, we performed chromatin assembly up to 15:1 mass ratio but were still unable to observe histone deposition (Figure S3E). Npm binds chromatin in the egg and DNA in vitro (Lu et al, 2012; Okuwaki et al, 2012). To exclude the possibility that the DNA binding prevented eNpm histone deposition, we performed a native gel shift assay using Npm and a linear DNA fragment.…”

Section: Resultsmentioning

confidence: 99%

Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

Onikubo

Nicklay

et al. 2015

Cell Reports

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Nucleoplasmin (Npm) is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs) specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity towards the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction leading to histone sequestration in the egg.

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Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

Cited by 16 publications

References 45 publications

Importin β-dependent nuclear import of TopBP1 in ATR–Chk1 checkpoint in Xenopus egg extracts

Importin β-dependent nuclear import of TopBP1 in ATR–Chk1 checkpoint in Xenopus egg extracts

Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation

Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

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