scholarly journals The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote mitotic NPC assembly

2021 ◽  
Author(s):  
Guillaume Holzer ◽  
Paola De Magistris ◽  
Cathrin Gramminger ◽  
Ruchika Sachdev ◽  
Adriana Magalska ◽  
...  
Keyword(s):  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Egg Extracts ◽  
Importin Α ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. We show here that Nup50 plays a crucial role in NPC assembly that is independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of the protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α, β, the GTPase Ran or chromatin is crucial for its function in the assembly process. Instead, we discovered that an N-terminal fragment of Nup50 can stimulate the Ran guanine exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in mitotic NPC reformation. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild type protein in in vitro NPC assembly assays.

Dimples, pores, star-rings, and thin rings on growing nuclear envelopes: evidence for structural intermediates in nuclear pore complex assembly

1997 ◽  
Vol 110 (4) ◽  
pp. 409-420 ◽  
Author(s):  
M.W. Goldberg ◽  
C. Wiese ◽  
T.D. Allen ◽  
K.L. Wilson
Keyword(s):  
Nuclear Pore Complex ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Complex Assembly ◽  
Egg Extracts ◽  
High Concentrations ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear envelopes in Xenopus egg extracts. Scattered among nuclear pore complexes were rare ‘dimples’ (outer membrane depressions, 5–35 nm diameter), more abundant holes (pores) with a variety of edge geometries (35–45 nm diameter; 3.3% of structures), pores containing one to eight triangular ‘star-ring’ subunits (2.1% of total), and more complicated structures. Neither mature complexes, nor these novel structures, formed when wheat germ agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (>500 microg/ml) directly to the assembly reaction; low concentrations (10 microg/ml) had no effect. However at intermediate concentrations (50–100 microg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of ‘empty’ pores, and other structures; this effect correlated with the lectin-induced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-binding nucleoporin. Another inhibitor, dibromo-BAPTA (5,5′-dibromo-1,2-bis[o-aminophenoxylethane-N,N,N′,N′-tetraacetic acid), had different effects depending on its time of addition to the assembly reaction. When 1 mM dibromo-BAPTA was added at time zero, no pore-related structures formed. However, when dibromo-BAPTA was added to growing nuclei 40–45 minutes after initiating assembly, star-rings and other structures accumulated, suggesting that dibromo-BAPTA can inhibit multiple stages in pore complex assembly. We propose that assembly begins with the formation and stabilization of a hole (pore) through the nuclear envelope, and that dimples, pores, star-rings, and thin rings are structural intermediates in nuclear pore complex assembly.

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent ‘envelope smoothing’ event

1997 ◽  
Vol 110 (13) ◽  
pp. 1489-1502 ◽  
Author(s):  
C. Wiese ◽  
M.W. Goldberg ◽  
T.D. Allen ◽  
K.L. Wilson
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Transmission Electron ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Attachment Proteins ◽  
Scanning Em ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying (‘rough’) vesicles, many of which bound directly to chromatin, and ‘smooth’ vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed ‘envelope smoothing’ occurred 5–15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that ‘smoothing’ required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

scholarly journals Nuclear Import of the Ran Exchange Factor, Rcc1, Is Mediated by at Least Two Distinct Mechanisms

2000 ◽  
Vol 149 (4) ◽  
pp. 835-850 ◽  
Author(s):  
Michael E. Nemergut ◽  
Ian G. Macara
Keyword(s):  
Nuclear Import ◽  
Nuclear Protein ◽  
Nucleotide Exchange ◽  
Ran Gtpase ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Interphase Cells ◽  
Importin Α

RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an ∼45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 into the nucleoplasm is extremely rapid. Import can proceed by at least two distinct mechanisms. The first is a classic import pathway mediated by basic residues within the NH2-terminal domain (NTD) of RCC1. This pathway is dependent upon both a preexisting Ran gradient and energy, and preferentially uses the importin-α3 isoform of importin-α. The second pathway is not mediated by the NTD of RCC1. This novel pathway does not require importin-α or importin-β or the addition of any other soluble factor in vitro; however, this pathway is saturable and sensitive only to a subset of inhibitors of classical import pathways. Furthermore, the nuclear import of RCC1 does not require a preexisting Ran gradient or energy. We speculate that this second import pathway evolved to ensure that RCC1 never accumulates in the cytoplasm.

scholarly journals Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

2008 ◽  
Vol 19 (4) ◽  
pp. 1753-1762 ◽  
Author(s):  
Lisa A. Hawryluk-Gara ◽  
Melpomeni Platani ◽  
Rachel Santarella ◽  
Richard W. Wozniak ◽  
Iain W. Mattaj
Keyword(s):  
Nuclear Envelope ◽  
Critical Role ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Egg Extracts ◽  
Multiple Copies ◽  
Xenopus Egg ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53–Nup155 complex plays a critical role in the processes of NPC and NE assembly.

scholarly journals TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts

2006 ◽  
Vol 173 (5) ◽  
pp. 685-694 ◽  
Author(s):  
Lori L. O'Brien ◽  
Christiane Wiese
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Envelope ◽  
Mitotic Spindle ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
Mitotic Spindle Assembly ◽  
Pore Complexes

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.

scholarly journals The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

1995 ◽  
Vol 6 (2) ◽  
pp. 227-236 ◽  
Author(s):  
J Rosenblatt ◽  
P Peluso ◽  
T J Mitchison
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Large Fraction ◽  
Nucleotide Exchange ◽  
Chromatography Analysis ◽  
Thymosin Beta 4 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

scholarly journals The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

2011 ◽  
Vol 193 (6) ◽  
pp. 1009-1020 ◽  
Author(s):  
Martijn Gloerich ◽  
Marjolein J. Vliem ◽  
Esther Prummel ◽  
Lars A.T. Meijer ◽  
Marije G.A. Rensen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Negative Regulator ◽  
Camp Signaling ◽  
Nuclear Pore ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Wide Range

Cyclic adenosine monophosphate (cAMP) is a second messenger that relays a wide range of hormone responses. In this paper, we demonstrate that the nuclear pore component RanBP2 acts as a negative regulator of cAMP signaling through Epac1, a cAMP-regulated guanine nucleotide exchange factor for Rap. We show that Epac1 directly interacts with the zinc fingers (ZNFs) of RanBP2, tethering Epac1 to the nuclear pore complex (NPC). RanBP2 inhibits the catalytic activity of Epac1 in vitro by binding to its catalytic CDC25 homology domain. Accordingly, cellular depletion of RanBP2 releases Epac1 from the NPC and enhances cAMP-induced Rap activation and cell adhesion. Epac1 also is released upon phosphorylation of the ZNFs of RanBP2, demonstrating that the interaction can be regulated by posttranslational modification. These results reveal a novel mechanism of Epac1 regulation and elucidate an unexpected link between the NPC and cAMP signaling.

scholarly journals Nucleus–Vacuole Junctions in Saccharomyces cerevisiae Are Formed Through the Direct Interaction of Vac8p with Nvj1p

2000 ◽  
Vol 11 (7) ◽  
pp. 2445-2457 ◽  
Author(s):  
Xiaozhou Pan ◽  
Paul Roberts ◽  
Yan Chen ◽  
Erik Kvam ◽  
Natalyia Shulga ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Fluorescent Protein ◽  
Close Contact ◽  
Direct Interaction ◽  
Integral Membrane Protein ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Importin Α ◽  
Green Fluorescent ◽  
Pore Complexes

Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including β-catenin and importin α, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus–vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40–60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in “orphan” rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Δ cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Δ andvac8-Δ cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.

scholarly journals Gef1p, a New Guanine Nucleotide Exchange Factor for Cdc42p, Regulates Polarity inSchizosaccharomyces pombe

2003 ◽  
Vol 14 (1) ◽  
pp. 313-323 ◽  
Author(s):  
Pedro M. Coll ◽  
Yadira Trillo ◽  
Amagoia Ametzazurra ◽  
Pilar Perez
Keyword(s):  
Cell Morphology ◽  
Rho Gtpases ◽  
Genetic Interaction ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Schizosaccharomyces pombe cdc42+regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1+increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression ofgef1+causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1+deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1Δ scd1Δ is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1+orscd1+causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.

scholarly journals RanBP2/Nup358 Provides a Major Binding Site for NXF1-p15 Dimers at the Nuclear Pore Complex and Functions in Nuclear mRNA Export

2004 ◽  
Vol 24 (3) ◽  
pp. 1155-1167 ◽  
Author(s):  
Daniel Forler ◽  
Gwénaël Rabut ◽  
Francesca D. Ciccarelli ◽  
Andrea Herold ◽  
Thomas Köcher ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Mrna Export ◽  
Stable Complex ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cytoplasmic Filaments ◽  
Nuclear Levels ◽  
Pore Complexes

ABSTRACT Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.

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