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.

scholarly journals Specialization of chromatin-bound nuclear pore complexes promotes yeast aging

2021 ◽  
Author(s):  
Anne C Meinema ◽  
Theo Aspert ◽  
Sung Sik Lee ◽  
Gilles Charvin ◽  
Yves Barral
Keyword(s):  
Quality Control ◽  
Nuclear Pore Complex ◽  
Mrna Export ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Replicative Lifespan ◽  
Yeast Aging ◽  
Key Drivers ◽  
Pore Complexes

The nuclear pore complex (NPC) mediates nearly all exchanges between nucleus and cytoplasm, and changes composition in many species as the organism ages. However, how these changes arise and whether they contribute themselves to aging is poorly understood. We show that in replicatively aging yeast cells attachment of DNA circles to NPCs drives the displacement of the NPCs’ nuclear basket and cytoplasmic complexes. Remodeling of the NPC resulted from the regulation of basket components by SAGA, rather than from damages. These changes affected NPC interaction with mRNA export factors, without affecting the residence of import factors or engaging the NPC quality control machinery. Mutations preventing NPC remodeling extended the replicative lifespan of the cells. Thus, our data indicate that DNA circles accumulating in the mother cell drive aging at least in part by triggering NPC specialization. We suggest that antagonistic pleiotropic effects of NPC specialization are key drivers of aging.

Defining the essential functional regions of the nucleoporin Nup145p

1997 ◽  
Vol 110 (7) ◽  
pp. 911-925 ◽  
Author(s):  
J.L. Emtage ◽  
M. Bucci ◽  
J.L. Watkins ◽  
S.R. Wente
Keyword(s):  
Amino Acid ◽  
Cleavage Site ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nuclear Pore Complexes ◽  
Permissive Temperature ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Pore Complexes

Studies of the essential nucleoporin Nup145p have shown that its depletion is coincident with a block in RNA export and that deletion of its amino-terminal domain results in clustering of nuclear pore complexes. To further define the functional domains of Nup145p, we have characterized a panel of nup145 mutants. Deletions from both the amino terminus and the carboxy terminus resulted in temperature sensitive mutants that accumulated polyadenylated RNA in the nucleus at the non-permissive temperature. In addition, these mutants also displayed constitutive clustering of nuclear pore complexes in localized patches of the nuclear envelope. These results suggested that an internal region of Nup145p consisting of amino acids 593–893 is essential for function. Accordingly, when this region was deleted, growth was not supported at any temperature, whereas the region alone was able to complement a null mutation when expressed on a high copy plasmid. Previous studies have suggested that Nup145p is cleaved into two polypeptides of approximately 65 and 80 kDa. Interestingly, our experiments suggest that cleavage occurs in vivo. However, a small internal deletion of 17 amino acid residues that abolished cleavage had no effect on cell growth. Therefore, cleavage is not necessary for Nup145p function. When a sequence harboring the Nup145p cleavage site required for Nup145p cleavage was inserted in a chimeric protein, it was not sufficient for mediating cleavage. Cleavage likely requires a second region from amino acid residues 247–524 in addition to the cleavage site.

The diverse roles of the Nup93/Nic96 complex proteins – structural scaffolds of the nuclear pore complex with additional cellular functions

2014 ◽  
Vol 395 (5) ◽  
pp. 515-528 ◽  
Author(s):  
Benjamin Vollmer ◽  
Wolfram Antonin
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Building Blocks ◽  
Transport Processes ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cellular Functions ◽  
Complex Assembly ◽  
Essential Function ◽  
Pore Complexes

Abstract Nuclear pore complexes mediate the transport between the cell nucleoplasm and cytoplasm. These 125 MDa structures are among the largest assemblies found in eukaryotes, built from proteins organized in distinct subcomplexes that act as building blocks during nuclear pore complex biogenesis. In this review, we focus on one of these subcomplexes, the Nup93 complex in metazoa and its yeast counterpart, the Nic96 complex. We discuss its essential function in nuclear pore complex assembly as a linker between the nuclear membrane and the central part of the pore and its various roles in nuclear transport processes and beyond.

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 In Vivo Dynamics of Nuclear Pore Complexes in Yeast

1997 ◽  
Vol 136 (6) ◽  
pp. 1185-1199 ◽  
Author(s):  
Mirella Bucci ◽  
Susan R. Wente
Keyword(s):  
Nuclear Envelope ◽  
Recipient Cell ◽  
Nucleocytoplasmic Transport ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Central Location ◽  
Nuclear Membranes ◽  
Mutant Strains ◽  
Pore Complexes

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.

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 Isolation of the yeast nuclear pore complex.

1993 ◽  
Vol 123 (4) ◽  
pp. 771-783 ◽  
Author(s):  
M P Rout ◽  
G Blobel
Keyword(s):  
Electron Microscopy ◽  
Image Reconstruction ◽  
Nuclear Pore Complex ◽  
Sedimentation Coefficient ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy ◽  
Pore Complexes

Nuclear pore complexes (NPCs) have been isolated from the yeast Saccharomyces. Negative stain electron microscopy of the isolated NPCs and subsequent image reconstruction revealed the octagonal symmetry and many of the ultrastructural features characteristic of vertebrate NPCs. The overall dimensions of the yeast NPC, both in its isolated form as well as in situ, are smaller than its vertebrate counterpart. However, the diameter of the central structures are similar. The isolated yeast NPC has a sedimentation coefficient of approximately 310 S and an M(r) of approximately 66 MD. It retains all but one of the eight known NPC proteins. In addition it contains as many as 80 uncharacterized proteins that are candidate NPC proteins.

scholarly journals Ensuring that yeast cells get their inheritance

2013 ◽  
Vol 203 (2) ◽  
pp. 167-167
Author(s):  
Mitch Leslie
Keyword(s):  
Daughter Cell ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Pore Complexes

A nucleoporin allows nuclear pore complexes access to daughter cell during mitosis.

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 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.

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