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 The Three Fungal Transmembrane Nuclear Pore Complex Proteins of Aspergillus nidulans Are Dispensable in the Presence of an Intact An-Nup84-120 Complex

2009 ◽  
Vol 20 (2) ◽  
pp. 616-630 ◽  
Author(s):  
Hui-Lin Liu ◽  
Colin P.C. De Souza ◽  
Aysha H. Osmani ◽  
Stephen A. Osmani
Keyword(s):  
High Temperature ◽  
Nuclear Envelope ◽  
Aspergillus Nidulans ◽  
Nuclear Pore Complex ◽  
Mitotic Spindle ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Spindle Pole Bodies ◽  
Pore Complexes

In Aspergillus nidulans nuclear pore complexes (NPCs) undergo partial mitotic disassembly such that 12 NPC proteins (Nups) form a core structure anchored across the nuclear envelope (NE). To investigate how the NPC core is maintained, we affinity purified the major core An-Nup84-120 complex and identified two new fungal Nups, An-Nup37 and An-ELYS, previously thought to be vertebrate specific. During mitosis the An-Nup84-120 complex locates to the NE and spindle pole bodies but, unlike vertebrate cells, does not concentrate at kinetochores. We find that mutants lacking individual An-Nup84-120 components are sensitive to the membrane destabilizer benzyl alcohol (BA) and high temperature. Although such mutants display no defects in mitotic spindle formation, they undergo mitotic specific disassembly of the NPC core and transient aggregation of the mitotic NE, suggesting the An-Nup84-120 complex might function with membrane. Supporting this, we show cells devoid of all known fungal transmembrane Nups (An-Ndc1, An-Pom152, and An-Pom34) are viable but that An-ndc1 deletion combined with deletion of individual An-Nup84-120 components is either lethal or causes sensitivity to treatments expected to destabilize membrane. Therefore, the An-Nup84-120 complex performs roles, perhaps at the NPC membrane as proposed previously, that become essential without the An-Ndc1 transmembrane Nup.

scholarly journals The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor.

1995 ◽  
Vol 131 (6) ◽  
pp. 1699-1713 ◽  
Author(s):  
M K Iovine ◽  
J L Watkins ◽  
S R Wente
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Structural Basis ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Repetitive Region ◽  
Amino Terminal ◽  
Section Electron ◽  
Rna Export

Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nup116p's role in NPC function. First, deletion mutagenesis analysis of NUP116 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nup100p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nup116p's GLFG region with either Nup100p's GLFG region or Nsp1p's FXFG repetitive region abolishes the function of Nup116p. At a semipermissive growth temperature, the cells lacking Nup116p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nup116p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin beta. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.

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 Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Jacqueline T. Brown ◽  
Alexandra J. Haraczy ◽  
Christopher M. Wilhelm ◽  
Kenneth D. Belanger
Keyword(s):  
Amino Acids ◽  
Nuclear Pore Complex ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Full Length ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Cytosolic Side ◽  
Carboxy Terminal ◽  
In Cells

AbstractPom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen that self-assembles to form the NPC membrane ring. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions of the protein are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in cells lacking endogenous Pom152 results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Targeted mutations in the amino-terminal and transmembrane domains did not alter Pom152 localization and neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.

scholarly journals Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae

Biology Open ◽  
2021 ◽  
Author(s):  
Jacqueline T. Brown ◽  
Alexandra J. Haraczy ◽  
Christopher M. Wilhelm ◽  
Kenneth D. Belanger
Keyword(s):  
Amino Acids ◽  
Nuclear Pore Complex ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Full Length ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Glycosylation Sites ◽  
Cytosolic Side ◽  
Carboxy Terminal

Pom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in pom152Δ cells results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.

NNF1 is an essential yeast gene required for proper spindle orientation, nucleolar and nuclear envelope structure and mRNA export

1997 ◽  
Vol 110 (14) ◽  
pp. 1615-1624 ◽  
Author(s):  
X. Shan ◽  
Z. Xue ◽  
G. Euskirchen ◽  
T. Melese
Keyword(s):  
Nuclear Envelope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Receptor Protein ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nuclear Pore Complexes ◽  
Yeast Gene ◽  
Localization Sequence

The nuclear envelope is central to nuclear structure and function. It plays a role in maintaining nuclear shape, allowing the exchange of macromolecules between the nucleus and the cytoplasm (via the nuclear pore complexes), and providing attachment sites for microtubules during chromosome segregation and nuclear migration (via the spindle pole body). We have isolated an essential yeast gene, NNF1 that is required for a number of nuclear functions. Cells depleted of Nnf1p or containing a temperature-sensitive nnf1 mutation have elongated microtubules and become bi- and multinucleate. They also have a fragmented nucleolous and accumulate poly(A)+ RNA inside the nucleus. A similar constellation of phenotypes has been reported in cells carrying mutations in a number of nuclear pore proteins, components of the Ran GTPase cycle, and the nuclear localization sequence receptor protein. Our results suggest that Nnf1p plays a role in a number of nuclear functions.

scholarly journals Nuclear Envelope Regulation of Oncogenic Processes: Roles in Pancreatic Cancer

Epigenomes ◽  
2018 ◽  
Vol 2 (3) ◽  
pp. 15 ◽  
Author(s):  
Claudia Preston ◽  
Randolph Faustino
Keyword(s):  
Pancreatic Cancer ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Structural Integrity ◽  
Radiation Treatment ◽  
Tumor Biology ◽  
Nuclear Lamina ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Linc Complex

Pancreatic cancer is an aggressive and intractable malignancy with high mortality. This is due in part to a high resistance to chemotherapeutics and radiation treatment conferred by diverse regulatory mechanisms. Among these, constituents of the nuclear envelope play a significant role in regulating oncogenesis and pancreatic tumor biology, and this review focuses on three specific components and their roles in cancer. The LINC complex is a nuclear envelope component formed by proteins with SUN and KASH domains that interact in the periplasmic space of the nuclear envelope. These interactions functionally and structurally couple the cytoskeleton to chromatin and facilitates gene regulation informed by cytoplasmic activity. Furthermore, cancer cell invasiveness is impacted by LINC complex biology. The nuclear lamina is adjacent to the inner nuclear membrane of the nuclear envelope and can actively regulate chromatin in addition to providing structural integrity to the nucleus. A disrupted lamina can impart biophysical compromise to nuclear structure and function, as well as form dysfunctional micronuclei that may lead to genomic instability and chromothripsis. In close relationship to the nuclear lamina is the nuclear pore complex, a large megadalton structure that spans both outer and inner membranes of the nuclear envelope. The nuclear pore complex mediates bidirectional nucleocytoplasmic transport and is comprised of specialized proteins called nucleoporins that are overexpressed in many cancers and are diagnostic markers for oncogenesis. Furthermore, recent demonstration of gene regulatory functions for discrete nucleoporins independent of their nuclear trafficking function suggests that these proteins may contribute more to malignant phenotypes beyond serving as biomarkers. The nuclear envelope is thus a complex, intricate regulator of cell signaling, with roles in pancreatic tumorigenesis and general oncogenic transformation.

scholarly journals A temperature-sensitive NUP116 null mutant forms a nuclear envelope seal over the yeast nuclear pore complex thereby blocking nucleocytoplasmic traffic.

1993 ◽  
Vol 123 (2) ◽  
pp. 275-284 ◽  
Author(s):  
S R Wente ◽  
G Blobel
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Nuclear Periphery ◽  
Dense Material ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Electron Microscopic ◽  
Electron Dense Material ◽  
Cytoplasmic Face ◽  
Delta Cells

NUP116 encodes a 116-kD yeast nuclear pore complex (NPC) protein that is not essential but its deletion (nup116 delta) slows cell growth at 23 degrees C and is lethal at 37 degrees C (Wente, S. R., M. P. Rout, and G. Blobel. 1992. J. Cell Biol. 119:705-723). Electron microscopic analysis of nup116 delta cells shifted to growth at 37 degrees C revealed striking perturbations of the nuclear envelope: a double membrane seal that was continuous with the inner and outer nuclear membranes had formed over the cytoplasmic face of the NPCs. Electron-dense material was observed accumulating between the cytoplasmic face of these NPCs and the membrane seal, resulting in "herniations" of the nuclear envelope around individual NPCs. In situ hybridization with poly(dT) probes showed the accumulation of polyadenylated RNA in the nuclei of arrested nup116 delta cells, sometimes in the form of punctate patches at the nuclear periphery. This is consistent with the electron microscopically observed accumulation of electron-dense material within the nuclear envelope herniations. We propose that nup116 delta NPCs remain competent for export, but that the formation of the membrane seals over the NPCs blocks nucleocytoplasmic traffic.

scholarly journals Expression of TorsinA in a heterologous yeast system reveals interactions with lumenal domains of LINC and nuclear pore complex components

2019 ◽  
Vol 30 (5) ◽  
pp. 530-541 ◽  
Author(s):  
Madeleine Chalfant ◽  
Karl W. Barber ◽  
Sapan Borah ◽  
David Thaller ◽  
C. Patrick Lusk
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Mammalian Cells ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Gene Encoding ◽  
Aaa Atpase ◽  
Dyt1 Dystonia ◽  
Physiological Relevance ◽  
Membrane Spanning

DYT1 dystonia is caused by an in-frame deletion of a glutamic acid codon in the gene encoding the AAA+ ATPase TorsinA (TorA). TorA localizes within the lumen of the nuclear envelope/endoplasmic reticulum and binds to a membrane-spanning cofactor, lamina associated polypeptide 1 (LAP1) or lumenal domain like LAP1 (LULL1), to form an ATPase; the substrate(s) of TorA remains ill-defined. Here we use budding yeast, which lack Torsins, to interrogate TorA function. We show that TorA accumulates at nuclear envelope-embedded spindle pole bodies (SPBs) in a way that requires its oligomerization and the SUN (Sad1 and UNc-84)-domain protein, Mps3. We further show that TorA physically interacts with human SUN1/2 within this system, supporting the physiological relevance of these interactions. Consistent with the idea that TorA acts on a SPB substrate, its binding to SPBs is modulated by the ATPase-stimulating activity of LAP1. TorA and TorA-ΔE reduce the fitness of cells expressing mps3 alleles, whereas TorA alone inhibits growth of cells lacking Pom152, a component of the nuclear pore complex. This genetic specificity is mirrored biochemically as TorA, but not TorA-ΔE, binds Pom152. Thus, TorA–nucleoporin interactions might be abrogated by TorA-ΔE, suggesting new experimental avenues to interrogate the molecular basis behind nuclear envelope herniations seen in mammalian cells lacking TorA function.

scholarly journals A new family of yeast nuclear pore complex proteins.

1992 ◽  
Vol 119 (4) ◽  
pp. 705-723 ◽  
Author(s):  
S R Wente ◽  
M P Rout ◽  
G Blobel
Keyword(s):  
Nuclear Pore Complex ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nuclear Pore Complexes ◽  
Amino Terminal ◽  
New Family ◽  
Fluorescence And Electron Microscopy ◽  
Pore Complexes ◽  
Modular Domains

We have identified a novel family of yeast nuclear pore complex proteins. Three individual members of this family, NUP49, NUP100, and NUP116, have been isolated and then characterized by a combination of molecular genetics and immunolocalization. Employing immunoelectron and immunofluorescence microscopy on yeast cells, we found that the binding of a polyspecific monoclonal antibody recognizing this family was predominantly at the nuclear pore complexes. Furthermore, the tagging of NUP49 with a unique epitope enabled the immunolocalization of this protein to the nuclear pore complex by both fluorescence and electron microscopy. DNA sequence analysis has shown that the amino-terminal regions of NUP49, NUP100, and NUP116 share repeated "GLFG" motifs separated from each other by glutamine, asparagine, serine and threonine rich spacers. All three proteins lack a repetitive domain found in the two precisely described yeast nuclear pore complex proteins. Only NUP49 is essential for cell viability. NUP116-deficient cells grow very slowly and are temperature sensitive, whereas the lack of NUP100 has no detectable phenotype. NUP100 and NUP116 are homologous over their entire lengths. Interestingly, NUP100 and NUP116 are both flanked by a histidine tRNA gene and a transposon element suggesting that they may have arisen by gene duplication. We propose that subfamilies of pore complex proteins can be defined by their characteristic combinations of different modular domains.

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