scholarly journals Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene.

1996 ◽  
Vol 7 (11) ◽  
pp. 1835-1855 ◽  
Author(s):  
C DeHoratius ◽  
P A Silver
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Temperature Sensitive Mutants ◽  
Nuclear Protein Import

To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein. We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants. We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein. The npl4-1 mutant was isolated by the previously described selection scheme. The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants. The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import. Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells. In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus. The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA. The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins. Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport.

scholarly journals O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import.

1992 ◽  
Vol 116 (2) ◽  
pp. 271-280 ◽  
Author(s):  
R Sterne-Marr ◽  
J M Blevitt ◽  
L Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Mammalian Cells ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Pore ◽  
Reticulocyte Lysate ◽  
Nuclear Protein Import ◽  
Cytosolic Factor ◽  
Cytosolic Factors

Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N-ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component.

scholarly journals Reconstitution of nuclear protein transport with semi-intact yeast cells.

1993 ◽  
Vol 123 (4) ◽  
pp. 785-798 ◽  
Author(s):  
G Schlenstedt ◽  
E Hurt ◽  
V Doye ◽  
P A Silver
Keyword(s):  
Nuclear Envelope ◽  
Protein Transport ◽  
Protein Import ◽  
Nuclear Protein ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Protein ◽  
Nuclear Protein Import ◽  
Pore Protein

We have developed an in vitro nuclear protein import reaction from semi-intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLS-containing proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nsp1 inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nsp1 mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined.

scholarly journals Sequence and characterization of cytoplasmic nuclear protein import factor p97.

1995 ◽  
Vol 130 (2) ◽  
pp. 265-274 ◽  
Author(s):  
N C Chi ◽  
E J Adam ◽  
S A Adam
Keyword(s):  
Nuclear Envelope ◽  
Metal Ion ◽  
Protein Import ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Nuclear Pore ◽  
Permeabilized Cell ◽  
Cytosolic Proteins ◽  
Cell Assay ◽  
Nuclear Protein Import

Nuclear location sequence-mediated binding of karyophilic proteins to the nuclear pore complexes is one of the earliest steps in nuclear protein import. We previously identified two cytosolic proteins that reconstitute this step in a permeabilized cell assay: the 54/56-kD NLS receptor and p97. A monoclonal antibody to p97 localizes the protein to the cytoplasm and the nuclear envelope. p97 is extracted from nuclear envelopes under the same conditions as the O-glycosylated nucleoporins indicating a tight association with the pore complex. The antibody inhibits import in a permeabilized cell assay but does not affect binding of karyophiles to the nuclear pore complex. Immunodepletion of p97 renders the cytosol inactive for import and identifies at least three other cytosolic proteins that interact with p97. cDNA cloning of p97 shows that it is a unique protein containing 23 cysteine residues. Recombinant p97 binds zinc and a bound metal ion is required for the nuclear envelope binding activity of the protein.

Abstract P217: Nuclear Protein Import as the Missing Link in Phenylephrine-Induced Cardiomyocyte Hypertrophy

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Mirna N Chahine ◽  
Maxime Mioulane ◽  
Gabor Földes ◽  
Alexander Lyon ◽  
Sian E Harding
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Translocation ◽  
Cardiomyocyte Hypertrophy ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
Adult Rat ◽  
Nuclear Protein Import

During cardiac hypertrophy, cardiomyocytes (CM) present alterations in gene expression and increased contractile protein content. Nuclear protein import (NPI) is critical in regulating gene expression, transcription, and subsequently cell hypertrophy. However, it is unknown how the nuclear transport machinery (transport receptors and nuclear pore complex (NPC)) functions to sustain increased demands for nucleocytoplasmic trafficking. The aim of this study was to determine if exposure of adult CM to phenylephrine (PE) affects hypertrophy by altering NPI and NPC density. Comparisons were made to adult failing rat and human CM. Rat myocytes were enzymatically isolated from adult hearts, and used for immunocytochemistry, qPCR and western immunoblotting. Failing CM were obtained from explanted human hearts at the time of transplant and from a rat model of myocardial infarction-induced hypertrophy and failure. Rat adult CM exposed for 48h to PE were injected with a protein import substrate (Alexa488-BSA-NLS) to visually monitor nuclear import with the confocal microscope. The effects of P38 MAPK inhibitor, HDAC inhibitor, Exportin-1 (CRM-1) inhibitor, and GSK-3 β inhibitor were investigated. Cell and nuclear sizes were increased in PE treated-adult rat CM and in the adult failing rat and human CM compared to normal CM. In contrast, PE depressed the rate and maximal NPI (by 65 +/- 3.4 % (3.55 from 5.46), p<0.05) as well as nucleoporin p62 mRNA and protein expression levels in adult rat CM compared to non-treated CM. Nucleoporin p62, cytoplasmic Ranbp1, and nuclear translocation of importins (Imp.α and β) relative densities were also decreased in PE treated-adult rat CM and in adult failing rat CM and human heart tissue compared to normal controls. On the contrary, CRM-1 nuclear export relative density was increased during the same pathological conditions. Thus NPI downregulation is linked to an increased nuclear export required by CM to generate the hypertrophic phenotype. All these effects were P38MAPK, HDAC and CRM-1 dependent but GSK-3Beta independent in rat CM. Our results show that alterations in NPI and NPC density occur in failing CM as well as in CM under hypertrophic stimuli. NPI may represent a critical therapeutic target in hypertrophic conditions.

scholarly journals nup1 mutants exhibit pleiotropic defects in nuclear pore complex function.

1994 ◽  
Vol 127 (2) ◽  
pp. 319-332 ◽  
Author(s):  
A M Bogerd ◽  
J A Hoffman ◽  
D C Amberg ◽  
G R Fink ◽  
L I Davis
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Structural Integrity ◽  
Mutational Analysis ◽  
Complex Function ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Amino Terminal ◽  
Carboxy Terminal

The NUP1 gene of Saccharomyces cerevisiae encodes one member of a family of nuclear pore complex proteins (nucleoporins) conserved from yeast to vertebrates. We have used mutational analysis to investigate the function of Nup1p. Deletion of either the amino- or carboxy-terminal domain confers a lethal phenotype, but partial truncations at either end affect growth to varying extents. Amino-terminal truncation causes mislocalization and degradation of the mutant protein, suggesting that this domain is required for targeting Nup1p to the nuclear pore complex. Carboxy-terminal mutants are stable but do not have wild-type function, and confer a temperature sensitive phenotype. Both import of nuclear proteins and export of poly(A) RNA are defective at the nonpermissive temperature. In addition, nup1 mutant cells become multinucleate at all temperatures, a phenotype suggestive of a defect in nuclear migration. Tubulin staining revealed that the mitotic spindle appears to be oriented randomly with respect to the bud, in spite of the presence of apparently normal cytoplasmic microtubules connecting one spindle pole body to the bud tip. EM analysis showed that the nuclear envelope forms long projections extending into the cytoplasm, which appear to have detached from the bulk of the nucleus. Our results suggest that Nup1p may be required to retain the structural integrity between the nuclear envelope and an underlying nuclear scaffold, and that this connection is required to allow reorientation of the nucleus in response to cytoskeletal forces.

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 GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

1996 ◽  
Vol 7 (12) ◽  
pp. 1921-1937 ◽  
Author(s):  
R Murphy ◽  
J L Watkins ◽  
S R Wente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Protein Import ◽  
Nuclear Transport ◽  
Complex Structure ◽  
Striking Similarity ◽  
Nuclear Pore ◽  
Pore Complexes

To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.

scholarly journals Nuclear envelope-vacuole contacts mitigate nuclear pore complex assembly stress

2020 ◽  
Author(s):  
Christopher L. Lord ◽  
Susan R. Wente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Lipid Droplets ◽  
Budding Yeast ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Cellular Mechanisms ◽  
Complex Assembly ◽  
Cellular Effects

AbstractThe intricacy of nuclear pore complex (NPC) biogenesis imposes risks of failure that can cause defects in nuclear transport and nuclear envelope morphology, however, cellular mechanisms utilized to alleviate NPC assembly stress are not well-defined. In the budding yeast Saccharomyces cerevisiae, we demonstrate that NVJ1- and MDM1-enriched nuclear envelope (NE)-vacuole contacts increase when NPC assembly is compromised in several nup mutants, including nup116ΔGLFG cells. These interorganelle nucleus-vacuole junctions (NVJs) cooperate with lipid droplets to maintain viability and enhance NPC formation in assembly mutants. Additionally, NVJs function with ATG1 to promote vacuole-dependent remodeling in nup116ΔGLFG cells, which also correlates with proper NPC formation. Importantly, NVJs significantly improve the physiology of NPC assembly mutants, despite having only negligible effects when NPC biogenesis is unperturbed. Collectively, these results define how NE-vacuole interorganelle contacts coordinate responses to mitigate deleterious cellular effects caused by disrupted NPC assembly.SummaryHow cells respond to deleterious effects imposed by disrupted nuclear pore complex (NPC) assembly are not well-defined. The authors demonstrate nuclear envelope-vacuole interactions expand in response to perturbed NPC assembly to promote viability, nuclear envelope remodeling, and proper NPC biogenesis.

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