Purification of a 60 kDa Nuclear Localization Signal Binding Protein in Rat Liver Nuclear Envelopes and Characterization of Its Properties

Haino, Makoto; Kawahire, Shigeru; Omata, Saburo; Horigome, Tsuneyoshi

doi:10.1093/oxfordjournals.jbchem.a124044

Cited by 9 publications

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“…(B) AL assembly reactions were incubated at room temperature for up to 3 h. At the designated times, an aliIt appeared from the data above that, upon incubation, the soluble nucleoporins became associated with the membrane fraction. When a similar A L assembly reaction w a s examined for the presence of nup58, which is found in a 600-kD complex with nup60 in the initial extract (Macaulay et al, 1995; see also Dabauvalle et al, 1990;Finlay et al, 1991;Haino et al, 1993), nup58 was also associated with the membrane pellet (Fig. 1 B).…”

Section: A Biochemical Assay For the Formation Of Annulate Lamellaementioning

confidence: 92%

Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation.

Meier

Miller

Forbes

1995

The Journal of Cell Biology

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Abstract. Formation of the nuclear pore is an intricate process involving membrane fusion and the ordered assembly of up to 1,000 pore proteins. As such, the study of pore assembly is not a simple one. Interestingly, annulate lamellae, a cytoplasmic organelle consisting of stacks of flattened membrane cisternae perforated by numerous pore complexes, have been found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvalle et al., 1991). In this work, a biochemical assay for annulate lamellae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into pore complexes in particular. Upon incubation of Xenopus egg cytosol and membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was time and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirmed that annulate lamellae were forming coincident with the incorporation of pore proteins into membranes. Homogenization and subsequent flotation of the membrane fraction allowed us to separate a population of dense membranes, containing the integral membrane pore protein gp210 and all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP~/S prevented incorporation of the soluble pore proteins into membranes. To address whether AL form in the absence of N-acetylglucosaminylated pore proteins, AL assembly was carried out in WGAsepharose--depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appearance. When the membrane fraction containing this altered AL was homogenized and subjected to flotation, the pore protein-containing membranes still sedimented in a distinct peak but were less dense than control annulate lamellae. T HE nuclear pore is responsible for establishing the distinct nuclear and cytoplasmic compartments of the eukaryotic cell. The pore is a highly selective channel through which macromolecular traffic must pass to enter or exit the nucleus. Although ions and other small molecules freely diffuse through the pore, larger molecules require a specific targeting signal for transit (for reviews, see Goldfarb and Michaud,.1991;Forbes, 1992;Gerace, 1992;Osborne and Silver, 1993;Powers and Forbes, 1994).The nuclear pore complex is a large and elaborate structure of ~120 million daltons and is comprised of N1,000 proteins. Structurally, it appears to consist of three ringsThe first two authors, Eva Meier and Brian R. Miller, contributed equally to the results presented here.Please address all correspondence to Douglass J. Forbes, Department of Biology, University of California at San Diego, La,lolla, CA 92093-0347. Tel.: (619) 534-0555....

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Section: A Biochemical Assay For the Formation Of Annulate Lamellaementioning

confidence: 92%

Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation.

Meier

Miller

Forbes

1995

The Journal of Cell Biology

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“…In chicken erythrocytes, an 18‐kDa membrane protein [18] and an LBR kinase were found to be associated with LBR [19]. LBR also bound a nuclear localization signal peptide [6,20], nucleoplasmin [6,20], and DNA [15] in vitro . It was shown by means of a two hybrid method that heterochromatin protein 1 (HP1) binds to LBR [21], and the binding site was localized to a region (residues 97–174) of the N‐terminal portion of human LBR [22].…”

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confidence: 99%

The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region

Takano¹,

Takeuchi²,

Ito

et al. 2002

European Journal of Biochemistry

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Binding of lamin B receptor (LBR) to chromatin was studied by means of an in vitro assay system involving recombinant fragments of human LBR and Xenopus sperm chromatin. Glutathione-S-transferase (GST)-fused proteins including LBR fragments containing the N-terminal region (residues 1-53) and arginine-serine repeat-containing region (residues 54-89) bound to chromatin. The binding of GST-fusion proteins incorporating the N-terminal and arginine-serine repeat-containing regions to chromatin was suppressed by mild trypsinization of the chromatin and by pretreatment with a DNA solution. A new cell-free system for analyzing the cell cycle-dependent binding of a protein to chromatin was developed from recombinant proteins, a Xenopus egg cytosol fraction and sperm chromatin. The system was applied to analyse the binding of LBR to chromatin. It was shown that the binding of LBR fragments to chromatin was stimulated by phosphorylation in the arginine-serine repeatcontaining region by a protein kinase(s) in a synthetic phase egg cytosol. However, the binding of LBR fragments was suppressed by phosphorylation at different residues in the same region by a kinase(s) in a mitotic phase cytosol. These results suggested that the cell cycle-dependent binding of LBR to chromatin is regulated by phosphorylation in the arginine-serine repeat-containing region by multiple kinases.Keywords: chromatin binding; lamin B receptor; LBR; Xenopus egg extract.The mature eggs of most vertebrates stay at the metaphase of the second meiotic division until they meet sperm. In that phase, the nuclear envelope is dispersed in the cytoplasm as nuclear envelope precursor vesicles. Cell cycle progression is triggered by fertilization, the nuclear envelope of the pronucleus being formed first. Then, the formation and disruption of nuclear envelopes occurs repeatedly during cleavage and in further differentiated somatic cell divisions. Thus, the structure of nuclear envelopes changes very dynamically depending on the stage of the cell cycle. To ensure the precise assembly/disassembly of nuclear envelopes in the cell cycle, the binding of proteins on nuclear envelope precursor vesicles/inner nuclear membranes to chromatin should be precisely regulated.Major nuclear envelope proteins known to bind to chromatin are lamins [1-4], lamin B receptor (LBR) [5,6], and LAP2b [7,8]. A peripheral nuclear membrane protein, Ya, is also known as a chromatin binding protein in early embryos of Drosophila melanogaster [9]. LAP2 was found as lamina-associated polypeptides in rat liver nuclear envelopes and shown to bind to chromatin at the N-terminal region [7,8]. It was shown recently that when a recombinant fragment of the protein was added to cell-free Xenopus egg nuclear assembly reactions at high concentrations, membrane binding to chromatin is inhibited [10]. LBR was found first as an avian erythrocyte-and liver-nuclear membrane protein [11,12]. Then, LBR was shown to be a chromatin-binding protein [5,6,13]. The segment two-thirds from the C-terminal of the LBR m...

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“…1) is only partly solubilized under these conditions. A similar combination of detergent and high salt has been used to extract a 180-kDa integral membrane protein from postsynaptic densities (Apperson and Kennedy, 1995) and a 60-kDa nuclear localization signal binding protein from nuclear envelopes (Haino et al ., 1993) . Attempts to purify detergent-solubilized PP59 on a variety of chromatographic matrices were disappointing .…”

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Characterization of the Phosphorylation Site of PP59, a Substrate for Cyclic AMP‐Dependent Protein Kinase That Is Enriched in the Synaptic Membrane Fraction of Rat Cerebellum

Rich

Aswad

1996

Journal of Neurochemistry

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We have identified previously a synaptic membrane‐associated protein, PP59, that serves as a substrate for cyclic AMP‐dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X‐100 plus 1 M KCl. A 290‐fold purification of PP59 was achieved by selective solubilization, followed by continuous‐elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP‐dependent protein kinase phosphorylation site within PP59, the partially purified 32P‐phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed‐phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala‐Arg‐Glu‐Arg‐Ser‐Asp‐Ser(P)‐Thr‐Gly‐Ser‐Ser‐Ser‐Val‐Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14‐amino acid sequence was used to develop polyclonal anti‐peptide antibodies that were affinity‐purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP‐dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.

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Purification of a 60 kDa Nuclear Localization Signal Binding Protein in Rat Liver Nuclear Envelopes and Characterization of Its Properties

Cited by 9 publications

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Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation.

Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation.

The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region

Characterization of the Phosphorylation Site of PP59, a Substrate for Cyclic AMP‐Dependent Protein Kinase That Is Enriched in the Synaptic Membrane Fraction of Rat Cerebellum

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