The Essential Yeast Nucleoporin NUP159 Is Located on the Cytoplasmic Side of the Nuclear Pore Complex and Serves in Karyopherin-mediated Binding of Transport Substrate

Kraemer, Doris; Strambio‐De‐Castillia, Caterina; Blobel, Günter; Rout, Michael P.

doi:10.1074/jbc.270.32.19017

Cited by 131 publications

(149 citation statements)

References 30 publications

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“…After trichloroacetic acid precipitation, the proteins were separated by SDS͞PAGE, transferred to poly-(vinylidene difluoride) membrane, and stained with amido black. A 43-kDa band was prepared for peptide sequencing as described (13,14). One sequence (YIFLRNAAEELK) matched that deduced from a partial human cDNA clone in GenBank (accession nos.…”

Section: Methodsmentioning

confidence: 99%

mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm

Kraemer

Blobel

1997

Proc. Natl. Acad. Sci. U.S.A.

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We have identified and molecularly characterized a human protein with a M r of 40,880 Da. After UV irradiation of HeLa cells, this protein was cross-linked to poly(A)-containing mRNA and was therefore designated mrnp 41 (for mRNA binding protein of 41 kDa). Cell fractionation and immunoblotting showed mrnp 41 in both the cytoplasm and the nucleus and particularly in the nuclear envelope. Immunof luorescence microscopy localized mrnp 41 to distinct foci in the nucleoplasm, to the nuclear rim, and to meshworklike structures throughout the cytoplasm. The cytoplasmic meshwork staining was disrupted by prior treatment of cells with the actin filament-or microtubule-disrupting drugs cytochalasin or nocodazole, respectively, suggesting association of mrnp 41 with the cytoskeleton. Double immunof luorescence with antibodies against mrnp 41 and the cytoplasmic poly(A) binding protein showed colocalization to the cytoplasmic meshwork. Immunogold electronmicroscopy confirmed mrnp 41's cytoplasmic and nucleoplasmic localization and revealed a striking labeling of nuclear pore complexes. Together these data suggest that mrnp 41 may function in nuclear export of mRNPs and͞or in cytoplasmic transport on, or attachment to, the cytoskeleton. Consistent with a role of mrnp 41 in nuclear export are previous reports that mutations in homologs of mrnp 41 in Schizosaccharomyces pombe, designated Rae1p, or in Saccharomyces cerevisiae, designated Gle2p, result in mRNA accumulation in the nucleus although it is presently not known whether these homologs are mRNA binding proteins as well.During their transcription, capping, splicing, and polyadenylation, nuclear mRNAs associate with a large number of proteins. These associations are dynamically coordinated with respect to various stages of mRNA synthesis, processing, and subsequent transport into the cytoplasm (reviewed in ref. 1). Some proteins are dissociated before nuclear mRNPs are transported across the nuclear pore complex (NPC) and remain in the nucleus (2). Whereas many proteins are dissociated shortly after nuclear mRNP transport across the NPC and are reimported into the nucleus (3), at least one of the nuclear mRNA binding proteins has been shown to remain bound to cytoplasmic mRNA (4). After transport across the NPC and displacement of nuclear binding proteins, the cytoplasmic mRNAs acquire a new set of binding proteins of which the cytoplasmic poly(A) binding protein (cPABP) is an example (5).Using in situ hybridization with oligo(dT) probes, the bulk of cytoplasmic mRNA has been shown to be attached to cytoskeletal elements, mostly microfilaments or microtubules (6, 7). It is not known whether cytoplasmic mRNAs are directly attached to the cytoskeleton. More likely, attachment is via mRNA-associated proteins. In addition to cytoskeletal attachment, specific mRNAs can be highly sublocalized within the cytoplasm. Examples for such specific sublocalization have been described in oocytes and eggs, as well as in somatic cells. This highly specific cytoplasmic sublocaliza...

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Section: Methodsmentioning

confidence: 99%

mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm

Kraemer

Blobel

1997

Proc. Natl. Acad. Sci. U.S.A.

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“…Samples were then transferred to buffer A without sucrose containing 3.7% formaldehyde and fixed for 15 min, and then given two washes in buffer A without sucrose for 5 min. Samples were blocked for 20 min in 1% BSA, 20 mM Tris-HCl, then incubated (with shaking) for 60 min with either the rabbit anti-Nup116-C antibody (1:10 dilution) (raised against the C-terminal 60kD domain and specific for Nup116p by Western blots and immunoprecipitation and affinity purified against the cognate epitope) or mouse Mab against Nup159p (Mab 165C10; 1:50 - Kraemer et al ., 1995), or with buffer as a control, and washed twice for 5 min with 0.001% (wt/vol) Tween 20, 20 mM TrisHCl. The samples were incubated for 30 min with secondary gold-conjugated goat anti-rabbit or anti-mouse antibody (AuroProb TM EM GAR IgG G10; 10 nm in diameter; Amersham) in 0.2% BSA-20 mM .…”

Section: Preparation Of Samples For Immuno-fesemmentioning

confidence: 99%

Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments

Киселева

Allen

Rutherford

et al. 2004

Journal of Structural Biology

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The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. AbstractThe nuclear pore complex (NPC) controls transport of macromolecules across the nuclear envelope. It is large and complex but appears to consist of only ~30 different proteins despite its mass of >60 MDa. Vertebrate NPC structure has been analyzed by several methods giving a comprehensive architectural model. Despite our knowledge of yeast nucleoporins, structural data is more limited and suggests the basic organization is similar to vertebrates, but may lack some peripheral and other components. Using field emission scanning electron microscopy to probe NPC structure we found that the yeast, like higher eukaryotic, NPCs contain similar peripheral components. We can detect cytoplasmic rings and evidence of nucleoplasmic rings in yeasts. A filamentous basket is present on the nucleoplasmic face and evidence for cytoplasmic filaments is shown. We observe a central structure, possibly the transporter, that which may be linked to the cytoplasmic ring by internal filaments. Immuno-gold labeling suggests that Nup159p may be attached to the cytoplasmic ring, whereas Nup116p may be associated, partly, with the cytoplasmic filaments. Analysis of a Nup57p mutant suggested a role in maintaining the stability of cytoplasmic components of the NPC. We conclude that peripheral NPC components appear similar in yeasts compared to higher organisms and present a revised model for yeast NPC structural composition.4

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“…Fig. 1 summarizes the Nup159 domains, namely, six presumptive LC8 recognition motifs in tandem flanked by an N-terminal predicted ␤-propeller domain (18) followed by a predominantly unstructured Phe-Gly-rich segment (19) and a C-terminal coiled-coil domain (20).…”

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Multiple Recognition Motifs in Nucleoporin Nup159 Provide a Stable and Rigid Nup159-Dyn2 Assembly

Nyarko

Song

Nováček

et al. 2013

Journal of Biological Chemistry

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Background: Nucleoporin Nup159 has multiple recognition motifs for Dyn2, the yeast ortholog of LC8. Results: Nup159 is intrinsically disordered and binds Dyn2 cooperatively at five of the six recognition motifs. Conclusion: Initial binding aligns two Nup chains in a bivalent scaffold; motifs 5 and 6 underlie rigidity. Significance: Multiple recognition sites provide entropy/enthalpy balance to a stable yet entropically unfavorable rigid complex.

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The Essential Yeast Nucleoporin NUP159 Is Located on the Cytoplasmic Side of the Nuclear Pore Complex and Serves in Karyopherin-mediated Binding of Transport Substrate

Cited by 131 publications

References 30 publications

mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm

mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm

Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments

Multiple Recognition Motifs in Nucleoporin Nup159 Provide a Stable and Rigid Nup159-Dyn2 Assembly

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