The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of ∼30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.

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Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells

The Journal of Cell Biology ◽

10.1083/jcb.200101089 ◽

2001 ◽

Vol 154 (1) ◽

pp. 71-84 ◽

Cited By ~ 276

Author(s):

Nathalie Daigle ◽

Joël Beaudouin ◽

Lisa Hartnell ◽

Gabriela Imreh ◽

Einar Hallberg ◽

...

Keyword(s):

Mammalian Cells ◽

Fluorescent Protein ◽

Nuclear Lamina ◽

Nuclear Pore ◽

Global Changes ◽

Nuclear Pore Complexes ◽

Annulate Lamellae ◽

Lamin B1 ◽

Green Fluorescent ◽

Pore Complexes

The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121–green fluorescent protein (GFP) and GFP-Nup153, and GFP–lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

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

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2445 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2445-2457 ◽

Cited By ~ 192

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.

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A Novel Fluorescence-based Genetic Strategy Identifies Mutants ofSaccharomyces cerevisiaeDefective for Nuclear Pore Complex Assembly

Molecular Biology of the Cell ◽

10.1091/mbc.9.9.2439 ◽

1998 ◽

Vol 9 (9) ◽

pp. 2439-2461 ◽

Cited By ~ 38

Author(s):

Mirella Bucci ◽

Susan R. Wente

Keyword(s):

Fluorescent Protein ◽

Nuclear Pore ◽

Temperature Sensitive ◽

Nuclear Pore Complexes ◽

Specific Subset ◽

Powerful Approach ◽

Sensitive Allele ◽

Green Fluorescent ◽

Pore Complexes ◽

Carboxy Terminal

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutantnup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57pE17and wt Nup49p. Interestingly, thenup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.

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Nuclear Pore Complex Is Able to Transport Macromolecules with Diameters of ∼39 nm

Molecular Biology of the Cell ◽

10.1091/mbc.01-06-0308 ◽

2002 ◽

Vol 13 (2) ◽

pp. 425-434 ◽

Cited By ~ 520

Author(s):

Nelly Panté ◽

Michael Kann

Keyword(s):

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Receptor Complexes ◽

Physiological Relevance ◽

B Virus ◽

Cargo Receptor ◽

Bidirectional Transport ◽

Targeting Signal ◽

Pore Complexes ◽

Transport Of Macromolecules

Bidirectional transport of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complexes (NPCs) by a signal-mediated mechanism that is directed by targeting signals (NLSs) residing on the transported molecules or “cargoes.” Nuclear transport starts after interaction of the targeting signal with soluble cellular receptors. After the formation of the cargo-receptor complex in the cytosol, this complex crosses the NPC. Herein, we use gold particles of various sizes coated with cargo-receptor complexes to determine precisely how large macromolecules crossing the NPC by the signal-mediated transport mechanism could be. We found that cargo-receptor-gold complexes with diameter close to 39 nm could be translocated by the NPC. This implies that macromolecules much larger than the assumed functional NPC diameter of 26 nm can be transported into the karyoplasm. The physiological relevance of this finding was supported by the observation that intact nucleocapsids of human hepatitis B virus with diameters of 32 and 36 nm are able to cross the nuclear pore without disassembly.

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Esc1, a Nuclear Periphery Protein Required for Sir4-Based Plasmid Anchoring and Partitioning

Molecular and Cellular Biology ◽

10.1128/mcb.22.23.8292-8301.2002 ◽

2002 ◽

Vol 22 (23) ◽

pp. 8292-8301 ◽

Cited By ~ 93

Author(s):

Erik D. Andrulis ◽

David C. Zappulla ◽

Athar Ansari ◽

Severine Perrod ◽

Catherine V. Laiosa ◽

...

Keyword(s):

Fluorescent Protein ◽

Nuclear Periphery ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Uncharacterized Protein ◽

Telomeric Silencing ◽

Green Fluorescent ◽

Pore Complexes ◽

Redundant Pathway

ABSTRACT A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Δesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Δesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.

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Pml39, a Novel Protein of the Nuclear Periphery Required for Nuclear Retention of Improper Messenger Ribonucleoparticles

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0527 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5258-5268 ◽

Cited By ~ 49

Author(s):

Benoît Palancade ◽

Michela Zuccolo ◽

Sophie Loeillet ◽

Alain Nicolas ◽

Valérie Doye

Keyword(s):

Fluorescent Protein ◽

Specific Interaction ◽

Nuclear Periphery ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nuclear Retention ◽

Reading Frame ◽

Nuclear Domains ◽

Green Fluorescent ◽

Pore Complexes

Using a genetic screen, we have identified a previously uncharacterized Saccharomyces cerevisiae open reading frame (renamed PML39) that displays a specific interaction with nucleoporins of the Nup84 complex. Localization of a Pml39-green fluorescent protein (GFP) fusion and two-hybrid studies revealed that Pml39 is mainly docked to a subset of nuclear pore complexes opposite to the nucleolus through interactions with Mlp1 and Mlp2. The absence of Pml39 leads to a specific leakage of unspliced mRNAs that is not enhanced upon MLP1 deletion. In addition, overexpression of PML39-GFP induces a specific trapping of mRNAs transcribed from an intron-containing reporter and of the heterogenous nuclear ribonucleoprotein Nab2 within discrete nuclear domains. In a nup60Δ mutant, Pml39 is mislocalized together with Mlp1 and Mlp2 in intranuclear foci that also recruit Nab2. Moreover, pml39Δ partially rescues the thermosensitive phenotypes of messenger ribonucleoparticles (mRNPs) assembly mutants, indicating that PML39 deletion also bypasses the requirement for normally assembled mRNPs. Together, these data indicate that Pml39 is an upstream effector of the Mlps, involved in the retention of improper mRNPs in the nucleus before their export.

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A nuclear pore sub-complex restricts the propagation of Ty retrotransposons by limiting their transcription

PLoS Genetics ◽

10.1371/journal.pgen.1009889 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1009889

Author(s):

Amandine Bonnet ◽

Carole Chaput ◽

Noé Palmic ◽

Benoit Palancade ◽

Pascale Lesage

Keyword(s):

Transcriptional Control ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Ltr Retrotransposons ◽

Loss Of Function ◽

Protein Coding ◽

Multiple Copies ◽

Ty1 Retrotransposition ◽

Pore Complexes ◽

The Impact

Beyond their canonical function in nucleocytoplasmic exchanges, nuclear pore complexes (NPCs) regulate the expression of protein-coding genes. Here, we have implemented transcriptomic and molecular methods to specifically address the impact of the NPC on retroelements, which are present in multiple copies in genomes. We report a novel function for the Nup84 complex, a core NPC building block, in specifically restricting the transcription of LTR-retrotransposons in yeast. Nup84 complex-dependent repression impacts both Copia and Gypsy Ty LTR-retrotransposons, all over the S. cerevisiae genome. Mechanistically, the Nup84 complex restricts the transcription of Ty1, the most active yeast retrotransposon, through the tethering of the SUMO-deconjugating enzyme Ulp1 to NPCs. Strikingly, the modest accumulation of Ty1 RNAs caused by Nup84 complex loss-of-function is sufficient to trigger an important increase of Ty1 cDNA levels, resulting in massive Ty1 retrotransposition. Altogether, our study expands our understanding of the complex interactions between retrotransposons and the NPC, and highlights the importance for the cells to keep retrotransposon under tight transcriptional control.

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Transport into and out of the Nucleus

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.65.4.570-594.2001 ◽

2001 ◽

Vol 65 (4) ◽

pp. 570-594 ◽

Cited By ~ 608

Author(s):

Ian G. Macara

Keyword(s):

Rna Processing ◽

Nucleocytoplasmic Transport ◽

Small Gtpase ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Facilitated Diffusion ◽

Transport Mechanisms ◽

Pore Complexes ◽

Transport Of Macromolecules ◽

Gtpase Ran

SUMMARY A defining characteristic of eukaryotic cells is the possession of a nuclear envelope. Transport of macromolecules between the nuclear and cytoplasmic compartments occurs through nuclear pore complexes that span the double membrane of this envelope. The molecular basis for transport has been revealed only within the last few years. The transport mechanism lacks motors and pumps and instead operates by a process of facilitated diffusion of soluble carrier proteins, in which vectoriality is provided by compartment-specific assembly and disassembly of cargo-carrier complexes. The carriers recognize localization signals on the cargo and can bind to pore proteins. They also bind a small GTPase, Ran, whose GTP-bound form is predominantly nuclear. Ran-GTP dissociates import carriers from their cargo and promotes the assembly of export carriers with cargo. The ongoing discovery of numerous carriers, Ran-independent transport mechanisms, and cofactors highlights the complexity of the nuclear transport process. Multiple regulatory mechanisms are also being identified that control cargo-carrier interactions. Circadian rhythms, cell cycle, transcription, RNA processing, and signal transduction are all regulated at the level of nucleocytoplasmic transport. This review focuses on recent discoveries in the field, with an emphasis on the carriers and cofactors involved in transport and on possible mechanisms for movement through the nuclear pores.

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Lumenal interactions in nuclear pore complex assembly and stability

Molecular Biology of the Cell ◽

10.1091/mbc.e10-06-0554 ◽

2011 ◽

Vol 22 (8) ◽

pp. 1375-1388 ◽

Cited By ~ 48

Author(s):

William T. Yewdell ◽

Paolo Colombi ◽

Taras Makhnevych ◽

C. Patrick Lusk

Keyword(s):

Genetic Interaction ◽

Nuclear Pore ◽

Physical Interaction ◽

Nuclear Pore Complexes ◽

Growth Defects ◽

Composition And Structure ◽

Genes Encoding ◽

Pore Complexes ◽

Lumenal Domain ◽

Transport Of Macromolecules

Nuclear pore complexes (NPCs) provide a gateway for the selective transport of macromolecules across the nuclear envelope (NE). Although we have a solid understanding of NPC composition and structure, we do not have a clear grasp of the mechanism of NPC assembly. Here, we demonstrate specific defects in nucleoporin distribution in strains lacking Heh1p and Heh2p—two conserved members of the LEM (Lap2, emerin, MAN1) family of integral inner nuclear membrane proteins. These effects on nucleoporin localization are likely of functional importance as we have defined specific genetic interaction networks between HEH1 and HEH2, and genes encoding nucleoporins in the membrane, inner, and outer ring complexes of the NPC. Interestingly, expression of a domain of Heh1p that resides in the NE lumen is sufficient to suppress both the nucleoporin mislocalization and growth defects in heh1Δpom34Δ strains. We further demonstrate a specific physical interaction between the Heh1p lumenal domain and the massive cadherin-like lumenal domain of the membrane nucleoporin Pom152p. These findings support a role for Heh1p in the assembly or stability of the NPC, potentially through the formation of a lumenal bridge with Pom152p.

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Members of the RSC Chromatin-Remodeling Complex Are Required for Maintaining Proper Nuclear Envelope Structure and Pore Complex Localization

Molecular Biology of the Cell ◽

10.1091/mbc.e09-07-0615 ◽

2010 ◽

Vol 21 (6) ◽

pp. 1072-1087 ◽

Cited By ~ 33

Author(s):

Laura C. Titus ◽

T. Renee Dawson ◽

Deborah J. Rexer ◽

Kathryn J. Ryan ◽

Susan R. Wente

Keyword(s):

Nuclear Envelope ◽

Chromatin Remodeling ◽

Fluorescent Protein ◽

Nuclear Periphery ◽

Structural Defects ◽

Nuclear Pore ◽

Temperature Sensitive ◽

Nuclear Pore Complexes ◽

Chromatin Remodeling Complex ◽

Green Fluorescent

The assembly, distribution, and functional integrity of nuclear pore complexes (NPCs) in the nuclear envelope (NE) are key determinants in the nuclear periphery architecture. However, the mechanisms controlling proper NPC and NE structure are not fully defined. We used two different genetic screening approaches to identify Saccharomyces cerevisiae mutants with defects in NPC localization. The first approach examined green fluorescent protein (GFP)-Nic96 in 531 strains from the yeast Tet-promoters Hughes Collection with individual essential genes expressed from a doxycycline-regulated promoter (TetO7-orf). Under repressive conditions, depletion of the protein encoded by 44 TetO7-orf strains resulted in mislocalized GFP-Nic96. These included STH1, RSC4, RSC8, RSC9, RSC58, ARP7, and ARP9, each encoding components of the RSC chromatin remodeling complex. Second, a temperature-sensitive sth1-F793S (npa18-1) mutant was identified in an independent genetic screen for NPC assembly (npa) mutants. NPC mislocalization in the RSC mutants required new protein synthesis and ongoing transcription, confirming that lack of global transcription did not underlie the phenotypes. Electron microscopy studies showed significantly altered NEs and nuclear morphology, with coincident cytoplasmic membrane sheet accumulation. Strikingly, increasing membrane fluidity with benzyl alcohol treatment prevented the sth1-F793S NE structural defects and NPC mislocalization. We speculate that NE structure is functionally linked to proper chromatin architecture.

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