Direct and indirect association of the small GTPase ran with nuclear pore proteins and soluble transport factors: studies in Xenopus laevis egg extracts.

Saitoh, Hisato; Cooke, Carol; Burgess, Wilson H.; Earnshaw, William C.; Dasso, Mary

doi:10.1091/mbc.7.9.1319

Cited by 61 publications

(64 citation statements)

References 60 publications

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“…Immunoprecipitation and immunoblotting were performed following standard procedures. Antibodies used were obtained as follows: anti-GST (a kind gift from Dr. Sarah Guadagno); anti-Xenopus Nup358 (Saitoh et al, 1996); monoclonal antibody 414, which recognizes Nup62, Nup153, Nup214, and Nup358 (Covance, Richmond, CA); HRP secondary antibodies (Zymed Laboratories, South San Francisco, CA); and a second antibody (Nup358 -2) against human Nup358 amino acids 2560 -2971 (Joseph et al, 2004). Antibodies against ␤-COP and the zinc finger region of Nup153 were generated as previously described (Liu et al, 2003).…”

Section: Immunoprecipitation and Immunoblottingmentioning

confidence: 99%

Nuclear Envelope Breakdown Is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

Prunuske

Liu

Elgort

et al. 2006

MBoC

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When higher eukaryotic cells transition into mitosis, the nuclear envelope, nuclear pore complexes, and nuclear lamina are coordinately disassembled. The COPI coatomer complex, which plays a major role in membrane remodeling at the Golgi, has been implicated in the process of nuclear envelope breakdown and requires interactions at the nuclear pore complex for recruitment to this new site of action at mitosis. Nup153, a resident of the nuclear pore basket, was found to be involved in COPI recruitment, but the molecular nature of the interface between COPI and the nuclear pore has not been fully elucidated. To better understand what occurs at the nuclear pore at this juncture, we have probed the role of the nucleoporin Nup358/RanBP2. Nup358 contains a repetitive zinc finger domain with overall organization similar to a region within Nup153 that is critical to COPI association, yet inspection of these two zinc finger domains reveals features that also clearly distinguish them. Here, we found that the Nup358 zinc finger domain, but not a zinc finger domain from an unrelated protein, binds to COPI and dominantly inhibits progression of nuclear envelope breakdown in an assay that robustly recapitulates this process in vitro. Moreover, the Nup358 zinc finger domain interferes with COPI recruitment to the nuclear rim. Consistent with a role for this pore protein in coordinating nuclear envelope breakdown, Nup358-specific antibodies impair nuclear disassembly. Significantly, targeting either Nup153 or Nup358 for inhibition perturbs nuclear envelope breakdown, supporting a model in which these nucleoporins play nonredundant roles, perhaps contributing to COPI recruitment platforms on both the nuclear and cytoplasmic faces of the pore. We found that an individual zinc finger is the minimal interface for COPI association, although tandem zinc fingers are optimal. These results provide new information about the critical components of nuclear membrane remodeling and lay the foundation for a better understanding of how this process is regulated. INTRODUCTIONThe nuclear envelope creates a barrier that is critical to maintenance of the environment in the nucleus, specialized to support transcription and DNA replication. This double lipid membrane bilayer consists of an outer nuclear membrane, which is continuous with the endoplasmic reticulum (ER), and an inner nuclear membrane, which contains at least 78 different proteins, anchored via protein-protein interactions to the underlying nuclear lamina and chromatin . The nuclear envelope is perforated by nuclear pores, through which communication between the cytoplasm and nucleus occurs (Suntharalingam and Wente, 2003;Weis, 2003;Fahrenkrog et al., 2004;Pante, 2004). Despite the large size of the macromolecular nuclear pore complex (125 MDa in vertebrates), it is comprised of only ϳ30 different proteins, or nucleoporins (Nups), each present in multiple copies (Rout et al., 2000;Cronshaw et al., 2002). In metazoan cells, all these components-the lamina, nuclear pores, as well as the ...

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Section: Immunoprecipitation and Immunoblottingmentioning

confidence: 99%

Nuclear Envelope Breakdown Is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

Prunuske

Liu

Elgort

et al. 2006

MBoC

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“…p18 was prepared from egg extracts through coimmunoprecipitation using antip340 RanBP2 antibodies (16). Approximately 2 g of p18 was transferred to nitrocellulose membranes.…”

Section: Buffers and Reagentsmentioning

confidence: 99%

“…This construct (pHis 6 -UBC9) gives a protein with a 20-aa extension at the N terminus of Ubc9p, including a His tag. Expression of tagged Ubc9p was induced in Escherichia coli [strain BL21(DE3) pLysS] under standard conditions (16). His-tagged Ubc9p was purified on Ni ϩ2 -agarose (Novagen).…”

Section: Buffers and Reagentsmentioning

confidence: 99%

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RanBP2 associates with Ubc9p and a modified form of RanGAP1

Saitoh

Cavenagh

et al. 1997

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

181

161

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“…Extending from the cytoplasmic ring are eight short filaments (1,2,4,5). These filaments, composed in part of the nuclear pore proteins (or nucleoporins) Nup214/CAN and Nup358/RanBP2, contain the initial binding sights for nuclear import receptors and their cargo (6)(7)(8)(9)(10)(11)(12)(13)(14). Extending from the nuclear ring are eight thin filaments terminating in a smaller ring.…”

Section: Introductionmentioning

confidence: 99%

Purification of the Vertebrate Nuclear Pore Complex by Biochemical Criteria

Miller¹,

Forbes²

2000

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The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of ~120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin β, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals ~40-50 distinct protein bands. KeywordsNuclear pore; annulate lamellae; purification; nucleoporin; importin; vertebrate The regulated transport of macromolecules across the nuclear envelope is critical for the creation and maintenance of the discrete identities of the nucleus and cytosol. Traffic through the nuclear envelope utilizes the nuclear pore complex, a large proteinaceous structure in excess of 120 million daltons in mass (1-3). As such, the nuclear pore represents one of the largest biological machines within the cell. The pore itself consists of three stacked octagonally symmetric rings, with the central ring composed of spoke-like structures supporting a central transporter ( Figure 1B). Extending from the cytoplasmic ring are eight short filaments (1,2,4,5). These filaments, composed in part of the nuclear pore proteins (or nucleoporins) Nup214/CAN and Nup358/RanBP2, contain the initial binding sights for nuclear import receptors and their cargo (6)(7)(8)(9)(10)(11)(12)(13)(14). Extending from the nuclear ring are eight thin filaments terminating in a smaller ring. This nuclear pore 'basket' minimally contains the nucleoporins Nup93, Nup98, and Nup153, and plays roles both in export from the

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Direct and indirect association of the small GTPase ran with nuclear pore proteins and soluble transport factors: studies in Xenopus laevis egg extracts.

Cited by 61 publications

References 60 publications

Nuclear Envelope Breakdown Is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

Nuclear Envelope Breakdown Is Coordinated by Both Nup358/RanBP2 and Nup153, Two Nucleoporins with Zinc Finger Modules

RanBP2 associates with Ubc9p and a modified form of RanGAP1

Purification of the Vertebrate Nuclear Pore Complex by Biochemical Criteria

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