scholarly journals Nup214 Is Required for CRM1-Dependent Nuclear Protein Export In Vivo

2006 ◽  
Vol 26 (18) ◽  
pp. 6772-6785 ◽  
Author(s):  
Saskia Hutten ◽  
Ralph H. Kehlenbach
Keyword(s):  
Protein Import ◽  
Nuclear Protein ◽  
Biochemical Properties ◽  
Protein Export ◽  
Nuclear Pore ◽  
Minor Effect ◽  
Nuclear Pore Complexes ◽  
High Affinity ◽  
Pore Complexes

ABSTRACT Nucleoporins mediate transport of macromolecules across the nuclear pore complex, yet the function of many individual nucleoporins is largely unresolved. To address this question, we depleted cells of the cytoplasmic nucleoporins Nup214/CAN and Nup358/RanBP2 by RNA interference. Depletion of Nup214 resulted in codepletion of its binding partner, Nup88. Nuclear pore complexes assembled in the absence of Nup214/Nup88 or Nup358 were fully functional in nuclear protein import, whereas nuclear mRNA export was slightly impaired. Depletion of Nup358 had only a minor effect on nuclear protein export. In contrast, depletion of Nup214/Nup88 led to strongly reduced CRM1-mediated export of the shuttling transcription factor NFAT as well as a human immunodeficiency virus-Rev derivative. A specific role of Nup214 in protein export is furthered by the biochemical properties of a high-affinity complex containing Nup214, CRM1, RanGTP, and an export cargo. Our results show that the Nup214/Nup88 complex is required for efficient CRM1-mediated transport, supporting a model involving a high-affinity binding site for CRM1 at Nup214 in the terminal steps of export.

scholarly journals Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein.

1992 ◽  
Vol 116 (1) ◽  
pp. 15-30 ◽  
Author(s):  
U F Greber ◽  
L Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Protein Import ◽  
Nuclear Protein ◽  
Complex Structure ◽  
Passive Diffusion ◽  
Nuclear Pore ◽  
Transport Inhibition ◽  
Cell Progression ◽  
Pore Complexes

Gp210 is a major transmembrane glycoprotein associated with the nuclear pore complex that is suggested to be important for organizing pore complex architecture and assembly. A mouse monoclonal IgG directed against an epitope in the lumenal domain of rat gp210 was expressed in cultured rat cells by microinjection of mRNA prepared from a hybridoma cell line. The expressed IgG, which becomes assembled into a functional antibody in the lumen of the endoplasmic reticulum, bound to the nuclear envelope in vivo. Expression of anti-gp210 antibody in interphase cells specifically reduced approximately fourfold the mediated nuclear import of a microinjected nuclear protein (nucleoplasmin) coupled to gold particles. The antibody also significantly decreased nuclear influx of a 10-kD dextran by passive diffusion. This transport inhibition did not result from removal of pore complexes from nuclear membranes or from gross alterations in pore complex structure, as shown by EM and immunocytochemistry. A physiological consequence of this transport inhibition was inhibition of cell progression from G2 into M phase. Hence, binding of this antibody to the lumenal side of gp210 must have a transmembrane effect on the structure and functions of the pore complex. These data argue that gp210 is directly or indirectly connected to pore complex constituents involved in mediated import and passive diffusion.

scholarly journals Caenorhabditis elegans Nucleoporins Nup93 and Nup205 Determine the Limit of Nuclear Pore Complex Size Exclusion In Vivo

2003 ◽  
Vol 14 (12) ◽  
pp. 5104-5115 ◽  
Author(s):  
Vincent Galy ◽  
Iain W. Mattaj ◽  
Peter Askjaer
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Envelope ◽  
Protein Import ◽  
Chromatin Condensation ◽  
Size Exclusion ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
C Elegans ◽  
Pore Complexes

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate communication between the nucleus and the cytoplasm. To obtain insight into the structure and function of NPCs of multicellular organisms, we have initiated an extensive analysis of Caenorhabditis elegans nucleoporins. Of 20 assigned C. elegans nucleoporin genes, 17 were found to be essential for embryonic development either alone or in combination. In several cases, depletion of nucleoporins by RNAi caused severe defects in nuclear appearance. More specifically, the C. elegans homologs of vertebrate Nup93 and Nup205 were each found to be required for normal NPC distribution in the nuclear envelope in vivo. Depletion of Nup93 or Nup205 caused a failure in nuclear exclusion of nonnuclear macromolecules of ∼70 kDa without preventing active nuclear protein import or the assembly of the nuclear envelope. The defects in NPC exclusion were accompanied by abnormal chromatin condensation and early embryonic arrest. Thus, the contribution to NPC structure of Nup93 and Nup205 is essential for establishment of normal NPC function and for cell viability.

scholarly journals Molecular mechanism of translocation through nuclear pore complexes during nuclear protein import

FEBS Letters ◽  
2001 ◽  
Vol 498 (2-3) ◽  
pp. 145-149 ◽  
Author(s):  
Murray Stewart ◽  
Rosanna P Baker ◽  
Richard Bayliss ◽  
Lesley Clayton ◽  
Richard P Grant ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Protein Import ◽  
Pore Complexes

Recombinant Nup153 Incorporates in Vivo into Xenopus Oocyte Nuclear Pore Complexes

2000 ◽  
Vol 129 (2-3) ◽  
pp. 306-312 ◽  
Author(s):  
Nelly Panté ◽  
Franziska Thomas ◽  
Ueli Aebi ◽  
Brian Burke ◽  
Ricardo Bastos
Keyword(s):  
Xenopus Oocyte ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Pore Complexes

scholarly journals A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sarah A. Nordeen ◽  
Kasper R. Andersen ◽  
Kevin E. Knockenhauer ◽  
Jessica R. Ingram ◽  
Hidde L. Ploegh ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Complex Structure ◽  
Constitutive Expression ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Modular Assembly ◽  
High Affinity ◽  
Ring Complex ◽  
Pore Complexes

AbstractNuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

scholarly journals Internuclear exchange of an inner nuclear membrane protein (p55) in heterokaryons: in vivo evidence for the interaction of p55 with the nuclear lamina.

1990 ◽  
Vol 111 (6) ◽  
pp. 2225-2234 ◽  
Author(s):  
L Powell ◽  
B Burke
Keyword(s):  
Nuclear Membrane ◽  
Lateral Diffusion ◽  
Nuclear Lamina ◽  
Membrane System ◽  
Mouse Cell ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Inner Nuclear Membrane ◽  
Pore Complexes

The movement between nuclei of an integral protein of the inner nuclear membrane has been studied in rat/mouse and rat/hamster heterokaryons. This protein, p55, was found to equilibrate between nuclei over a period of approximately 6 h in the absence of new protein synthesis. When rat/mouse heterokaryons were constructed using an undifferentiated murine embryonal carcinoma (P19), which lacks lamins A and C, no accumulation of p55 in the mouse cell nucleus was observed. However, P19 nuclei could be rendered competent to accumulate p55 by transfecting the parent cells with human lamin A before cell fusion, supporting the notion that p55 may interact with the nuclear lamina. Since p55 does not appear to be able to dissociate from the nuclear membrane, it is concluded that this exchange between nuclei does not occur in the aqueous phase and instead is probably membrane mediated. It is proposed that this protein may be free to move between the inner and outer nuclear membranes via the continuities at the nuclear pore complexes and that transfer between nuclei occurs via lateral diffusion through the peripheral ER, which appears to form a single continuous membrane system in these heterokaryons. One implication of these observations is that accumulation of at least some integral proteins in the inner nuclear membrane may be mediated by interactions with other nuclear components and may not require a single defined targeting sequence.

scholarly journals Targeting and function in mRNA export of nuclear pore complex protein Nup153.

1996 ◽  
Vol 134 (5) ◽  
pp. 1141-1156 ◽  
Author(s):  
R Bastos ◽  
A Lin ◽  
M Enarson ◽  
B Burke
Keyword(s):  
Nuclear Pore Complex ◽  
Protein Import ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Terminal Domain ◽  
General Decline ◽  
Complex Protein ◽  
Pore Complexes ◽  
And Function

Nup153 is a large (153 kD) O-linked glyco-protein which is a component of the basket structure located on the nucleoplasmic face of nuclear pore complexes. This protein exhibits a tripartite structure consisting of a zinc finger domain flanked by large (60-70 kD) NH2- and COOH-terminal domains. When full-length human Nup153 is expressed in BHK cells, it accumulates appropriately at the nucleoplasmic face of the nuclear envelope. Targeting information for Nup153 resides in the NH2-terminal domain since this region of the molecule can direct an ordinarily cytoplasmic protein, pyruvate kinase, to the nuclear face of the nuclear pore complex. Overexpression of Nup153 results in the dramatic accumulation of nuclear poly (A)+ RNA, suggesting an inhibition of RNA export from the nucleus. This is not due to a general decline in nucleocytoplasmic transport or to occlusion or loss of nuclear pore complexes since nuclear protein import is unaffected. While overexpression of certain Nup153 constructs was found to result in the formation of unusual intranuclear membrane arrays, this structural phenotype could not be correlated with the effects on poly (A)+ RNA distribution. The RNA trafficking defect was, however, dependent upon the Nup153 COOH-terminal domain which contains most of the XFXFG repeats. It is proposed that this region of Nup153, lying within the distal ring of the nuclear basket, represents a docking site for mRNA molecules exiting the nucleus.

scholarly journals Origin of the nuclear proteome on the basis of pre-existing nuclear localization signals in prokaryotic proteins

2020 ◽  
Author(s):  
Olga M. Lisitsyna ◽  
Margarita A. Kurnaeva ◽  
Eugene A. Arifulin ◽  
Maria Y. Shubina ◽  
Yana R. Musinova ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Protein Import ◽  
Eukaryotic Cell ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Localization Signals ◽  
Binding Domains ◽  
Nuclear Proteome ◽  
Pore Complexes ◽  
Open Question

AbstractThe origin of the selective nuclear protein import machinery, which consists of nuclear pore complexes and adaptor molecules interacting with the nuclear localization signals (NLSs) of cargo molecules, was one of the most important events in the evolution of the eukaryotic cell. How the proteins were selected for import into the forming nuclei remains an open question. Here, we demonstrate that functional NLSs may be integrated inside nucleotide-binding domains of both eukaryotic and prokaryotic proteins and may co-evolve with these domains. We propose that the pre-existence of NLSs inside prokaryotic proteins dictated, at least partially, the nuclear proteome composition.

scholarly journals NDC1 is necessary for stable assembly of the nuclear pore scaffold to establish nuclear transport in early C. elegans embryos

2021 ◽  
Author(s):  
Michael Sean Mauro ◽  
Gunta Celma ◽  
Vitaly Zimyanin ◽  
Kimberley H. Gibson ◽  
Stefanie Redemann ◽  
...  
Keyword(s):  
Outer Ring ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
3D Analysis ◽  
Protein Assemblies ◽  
C Elegans ◽  
Nuclear Assembly ◽  
Stable Association ◽  
Pore Complexes

Nuclear pore complexes (NPCs) are large protein assemblies that facilitate transport of macromolecules across the nuclear envelope (NE) [1, 2]. How thousands of NPCs rapidly assemble to form a functional NE after open mitosis is not known. Recruitment of the outer ring Nup107-160 complex to the NE initiates NPC assembly. The Nup53/93 complex bridges the outer ring to the central channel to form a functional pore [3-6]. Nup53 interacts with the conserved transmembrane nucleoporin Ndc1; however, how Ndc1 contributes to post-mitotic NPC assembly is unclear [7-9]. Here, we use C. elegans embryos to show that the timely formation of a functional NE after mitosis depends on Ndc1. Endogenously tagged Ndc1 is recruited early to the reforming NE and is highly mobile in the nuclear rim. 3D analysis of NE reformation revealed a significant decrease in NPC density in ndc1 deleted embryos: continuous nuclear membranes contained few holes where NPCs are normally located. Nup160 is highly mobile in NEs depleted of Ndc1 and outer ring scaffold components are less enriched at the rim. Nup160 is not recruited to the nuclear rim when both ndc1 and nup53 are absent and nuclear assembly fails. This suggests that Ndc1 and Nup53 function in part in parallel pathways to drive post-mitotic nuclear assembly in vivo. Together, we show that Ndc1 dynamically associates with the NE and promotes stable association of the outer ring scaffold with nascent NEs to facilitate NPC assembly after open mitosis, revealing a previously uncharacterized role for Ndc1 in forming functional NE.

scholarly journals Determination of the intracellular state of soluble macromolecules by gel filtration in vivo in the cytoplasm of amphibian oocytes.

1986 ◽  
Vol 102 (6) ◽  
pp. 2006-2014 ◽  
Author(s):  
M C Dabauvalle ◽  
W W Franke
Keyword(s):  
Light And Electron Microscopy ◽  
Gel Filtration ◽  
Size Exclusion ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Metabolic Labeling ◽  
Two Dimensional Gel Electrophoresis ◽  
Pore Complexes

A method to examine the diffusible state and the sizes of major cytoplasmic proteins in a living cell is described. Small (40-300 microns) commercially available gel filtration beads of a broad range of Mr exclusion limits were microsurgically implanted into the cytoplasm of oocytes of the frog, Xenopus laevis, usually after metabolic labeling of oocyte proteins with [35S]methionine. After equilibration in vivo for several hours, the appearance of the implanted cells, notably the bead-cytoplasm boundary, was examined by light and electron microscopy of sections and found to be unaffected. After incubation the beads were isolated, briefly rinsed, and their protein contents examined by one- or two-dimensional gel electrophoresis. We show that diffusible proteins can be identified by their inclusion in the pores of the gel filtration beads used and that their approximate sizes can be estimated from the size exclusion values of the specific materials used. The application of this method to important cell biological questions is demonstrated by showing that several "karyophobic proteins," i.e., proteins of the cytosolic fraction which accumulate in the cytoplasm in vivo, are indeed diffusible in the living oocyte and appear with sizes similar to those determined in vitro. This indicates that the nucleo-cytoplasmic distribution of certain diffusible proteins is governed, in addition to size exclusion at nuclear pore complexes and karyophilic "signals," by other, as yet unknown forces. Some possible applications of this method of gel filtration in vivo are discussed.

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