Identification and Characterization of Nuclear Pore Subcomplexes in Mitotic Extract of Human Somatic Cells

Matsuoka, Yosuke; Takagi, Motoki; Ban, Tadanobu; Miyazaki, Masao; Yamamoto, Takahiro; Kondo, Yukihiro; Yoneda, Yoshihiro

doi:10.1006/bbrc.1998.9953

Cited by 35 publications

(22 citation statements)

References 37 publications

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“…Several vertebrate nucleoporins form stable subcomplexes (e.g., Kita et al, 1993;Fornerod et al, 1997, Bastos et al, 1997, some of which also remain associated during mitosis (Grandi et al, 1997;Matsuoka et al, 1999;Miller et al, 2000;Belgareh et al, 2001;. In contrast, the mitotic form of Tpr does not remain bound to other nucleoporins, and extraction conditions required for releasing NPC-bound Tpr from interphase nuclei result in the release of soluble Tpr homodimers (our unpublished results; also Shah et al, 1998;Hase et al, 2001).…”

Section: In Vivo Cross-linking Confirms Proximity Between Presumptivementioning

confidence: 78%

Direct Interaction with Nup153 Mediates Binding of Tpr to the Periphery of the Nuclear Pore Complex

Hase

Cordes

2003

MBoC

168

212

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Tpr is a 267-kDa protein forming coiled coil-dominated homodimers that locate at the nucleoplasmic side of the nuclear pore complex (NPC). The proteins that tether Tpr to this location are unknown. Moreover, the question whether Tpr itself might act as a scaffold onto which other NPC components need to be assembled has not been answered to date. To assess Tpr's role as an architectural element of the NPC, we have studied the sequential disassembly and reassembly of NPCs in mitotic cells, paralleled by studies of cells depleted of Tpr as a result of posttranscriptional tpr gene silencing by RNA interference (RNAi). NPC assembly and recruitment of several nucleoporins, including Nup50, Nup93, Nup96, Nup98, Nup107, and Nup153, in anaphase/early telophase is shown to precede NPC association of Tpr in late telophase. In accordance, cellular depletion of Tpr by RNAi does not forestall binding of these nucleoporins to the NPC. In a search for proteins that moor Tpr to the NPC, we have combined the RNAi approach with affinity-chromatography and yeast two-hybrid interaction studies, leading to the identification of nucleoporin Nup153 as the binding partner for Tpr. The specificity of this interaction is demonstrated by its sensitivity to Tpr amino acid substitution mutations that abolish Tpr's ability to adhere to the NPC and affect the direct binding of Tpr to Nup153. Accordingly, cellular depletion of Nup153 by RNAi is shown to result in mislocalization of Tpr to the nuclear interior. Nup153 deficiency also causes mislocalization of Nup50 but has no direct effect on NPC localization of the other nucleoporins studied in this investigation. In summary, these results render Tpr a protein only peripherally attached to the NPC that does not act as an essential scaffold for other nucleoporins

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Section: In Vivo Cross-linking Confirms Proximity Between Presumptivementioning

confidence: 78%

Direct Interaction with Nup153 Mediates Binding of Tpr to the Periphery of the Nuclear Pore Complex

Hase

Cordes

2003

MBoC

168

212

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“…Such a self-interaction has been proposed for the initiation of Cajal body formation by p80 coilin (Hebert and Matera, 2000). Gel exclusion chromatography indicates that during mitosis Nup98 exists in a complex of ϳ450 kDa Matsuoka et al, 1999). The stoichiometry of this complex is unknown.…”

Section: Discussionmentioning

confidence: 99%

Nup98 Is a Mobile Nucleoporin with Transcription-dependent Dynamics

Griffis

Altan

Lippincott‐Schwartz

et al. 2002

MBoC

248

260

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Nucleoporin 98 (Nup98), a glycine-leucine-phenylalanine-glycine (GLFG) amino acid repeatcontaining nucleoporin, plays a critical part in nuclear trafficking. Injection of antibodies to Nup98 into the nucleus blocks the export of most RNAs. Nup98 contains binding sites for several transport factors; however, the mechanism by which this nucleoporin functions has remained unclear. Multiple subcellular localizations have been suggested for Nup98. Here we show that Nup98 is indeed found both at the nuclear pore complex and within the nucleus. Inside the nucleus, Nup98 associates with a novel nuclear structure that we term the GLFG body because the GLFG domain of Nup98 is required for targeting to this structure. Photobleaching of green fluorescent protein-Nup98 in living cells reveals that Nup98 is mobile and moves between these different localizations. The rate of recovery after photobleaching indicates that Nup98 interacts with other, less mobile, components in the nucleoplasm. Strikingly, given the previous link to nuclear export, the mobility of Nup98 within the nucleus and at the pore is dependent on ongoing transcription by RNA polymerases I and II. These data give rise to a model in which Nup98 aids in direction of RNAs to the nuclear pore and provide the first potential mechanism for the role of a mobile nucleoporin. INTRODUCTIONThe nuclear pore complex is a massive structure that conducts all traffic between the nucleus and cytoplasm (reviewed by Ohno et al., 1998;Gorlich and Kutay, 1999;Ryan and Wente, 2000;Vasu and Forbes, 2001). The pore has been studied intensely at the structural level in both yeast and vertebrate systems (Stoffler et al., 1999a;Allen et al., 2000). Although the vertebrate pore is larger and thought to contain a greater number of constituent proteins, the pores of both yeast and vertebrates share a similar structural organization. The central mass of the nuclear pore displays eightfold symmetry around a central axis perpendicular to the nuclear envelope. Two distinct sets of fibers extend out from the cytoplasmic and nuclear faces of the pore. On the nuclear side, the fibers are joined together at their distal ends by a ring to form the nuclear basket of the pore. Additionally, the nuclear basket of both yeast and vertebrate pores has associated filaments that extend for considerable distances into the nuclear interior and may serve to direct transport cargoes to and/or from the pore.A subset of the nuclear pore complex proteins (nucleoporins or Nups) each contain a domain with multiple, nontandem repeats of the amino acid sequences FG, FXFG, or GLFG (glycine-leucine-phenylalanine-glycine). These domains provide docking sites for a family of nuclear transport signal receptor proteins (known as importins, exportins, and transportin, or collectively as karyopherins). The repeat domain nucleoporins are found in multiple substructures of the nuclear pore and thus are thought to facilitate the moveArticle published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.01-11-0538. Article and p...

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“…Nup214 has been localized close to the midplane of the NE, possibly as a component of the cytoplasmic ring (34,64), and interacts with Nup88 to form a stable subcomplex (6,20,41). The mechanism for targeting this Nup88-Nup214 subcomplex to the NPC during nuclear assembly apparently requires both proteins, since depletion of Nup214 from mouse embryos caused mislocalization of Nup88 from the NPC, and the Nup214 interaction domain of Nup88 expressed in BHK cells mislocalized Nup214 to the cytoplasm (6,18,20).…”

mentioning

confidence: 99%

Nup358/RanBP2 Attaches to the Nuclear Pore Complex via Association with Nup88 and Nup214/CAN and Plays a Supporting Role in CRM1-Mediated Nuclear Protein Export

Bernad

Velde

Fornerod

et al. 2004

Molecular and Cellular Biology

155

170

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Nuclear pore complexes (NPCs) traverse the nuclear envelope (NE), providing a channel through which nucleocytoplasmic transport occurs. Nup358/RanBP2, Nup214/CAN, and Nup88 are components of the cytoplasmic face of the NPC. Here we show that Nup88 localizes midway between Nup358 and Nup214 and physically interacts with them. RNA interference of either Nup88 or Nup214 in human cells caused a strong reduction of Nup358 at the NE. Nup88 and Nup214 showed an interdependence at the NPC and were not affected by the absence of Nup358. These data indicate that Nup88 and Nup214 mediate the attachment of Nup358 to the NPC. We show that localization of the export receptor CRM1 at the cytoplasmic face of the NE is Nup358 dependent and represents its empty state. Also, removal of Nup358 causes a distinct reduction in nuclear export signal-dependent nuclear export. We propose that Nup358 provides both a platform for rapid disassembly of CRM1 export complexes and a binding site for empty CRM1 recycling into the nucleus.

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Identification and Characterization of Nuclear Pore Subcomplexes in Mitotic Extract of Human Somatic Cells

Cited by 35 publications

References 37 publications

Direct Interaction with Nup153 Mediates Binding of Tpr to the Periphery of the Nuclear Pore Complex

Direct Interaction with Nup153 Mediates Binding of Tpr to the Periphery of the Nuclear Pore Complex

Nup98 Is a Mobile Nucleoporin with Transcription-dependent Dynamics

Nup358/RanBP2 Attaches to the Nuclear Pore Complex via Association with Nup88 and Nup214/CAN and Plays a Supporting Role in CRM1-Mediated Nuclear Protein Export

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