scholarly journals Interference with the cytoplasmic tail of gp210 disrupts “close apposition” of nuclear membranes and blocks nuclear pore dilation

2002 ◽  
Vol 158 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Sheona P. Drummond ◽  
Katherine L. Wilson
Keyword(s):  
Pore Formation ◽  
Integral Membrane Protein ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Outer Membranes ◽  
Nuclear Assembly ◽  
Nuclear Membranes ◽  
Pore Dilation ◽  
Pore Complexes ◽  
Lumenal Domain

We tested the hypothesis that gp210, an integral membrane protein of nuclear pore complexes (NPCs), mediates nuclear pore formation. Gp210 has a large lumenal domain and small COOH-terminal tail exposed to the cytoplasm. We studied the exposed tail. We added recombinant tail polypeptides to Xenopus nuclear assembly extracts, or inhibited endogenous gp210 tails using anti-tail antibodies. Both strategies had no effect on the formation of fused flattened nuclear membranes, but blocked NPC assembly and nuclear growth. Inhibited nuclei accumulated gp210 and some nucleoporin p62, but failed to incorporate nup214/CAN, nup153, or nup98 and were defective for nuclear import of lamin B3. Scanning and transmission EM revealed a lack of “closely apposed” inner and outer membranes, and the accumulation of novel arrested structures including “mini-pores.” We conclude that gp210 has early roles in nuclear pore formation, and that pore dilation is mediated by gp210 and its tail-binding partner(s). We propose that membrane fusion and pore dilation are coupled, acting as a mechanism to control nuclear pore size.

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

2000 ◽  
Vol 11 (7) ◽  
pp. 2445-2457 ◽  
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.

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 Lumenal interactions in nuclear pore complex assembly and stability

2011 ◽  
Vol 22 (8) ◽  
pp. 1375-1388 ◽  
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.

scholarly journals The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex

2010 ◽  
Vol 189 (7) ◽  
pp. 1129-1142 ◽  
Author(s):  
Gandhi Theerthagiri ◽  
Nathalie Eisenhardt ◽  
Heinz Schwarz ◽  
Wolfram Antonin
Keyword(s):  
Membrane Proteins ◽  
Building Blocks ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Wild Type ◽  
Nuclear Assembly ◽  
Evolutionarily Conserved ◽  
Major Structural Component ◽  
Pore Complexes

All transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). Despite their enormous size, ∼60 MD in vertebrates, they are comprised of only ∼30 distinct proteins (nucleoporins or Nups), many of which form subcomplexes that act as building blocks for NPC assembly. One of these evolutionarily conserved subcomplexes, the Nup93 complex, is a major structural component linking the NPC to the membranes of the NE. Using in vitro nuclear assembly assays, we show that two components of the Nup93 complex, Nup188 and Nup205, are dispensable for NPC formation. However, nuclei lacking Nup188 increase in size by several fold compared with wild type. We demonstrate that this phenotype is caused by an accelerated translocation of integral membrane proteins through NPCs, suggesting that Nup188 confines the passage of membrane proteins and is thus crucial for the homeostasis of the different nuclear membranes.

scholarly journals The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes

2007 ◽  
Vol 178 (5) ◽  
pp. 799-812 ◽  
Author(s):  
John J. Scarcelli ◽  
Christine A. Hodge ◽  
Charles N. Cole
Keyword(s):  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Membrane Dynamics ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cold Sensitive ◽  
Low Levels ◽  
Lower Temperature ◽  
Pore Complexes ◽  
And Function

Although the structure and function of components of the nuclear pore complex (NPC) have been the focus of many studies, relatively little is known about NPC biogenesis. In this study, we report that Apq12 is required for efficient NPC biogenesis in Saccharomyces cerevisiae. Apq12 is an integral membrane protein of the nuclear envelope (NE) and endoplasmic reticulum. Cells lacking Apq12 are cold sensitive for growth, and a subset of their nucleoporins (Nups), those that are primarily components of the cytoplasmic fibrils of the NPC, mislocalize to the cytoplasm. APQ12 deletion also causes defects in NE morphology. In the absence of Apq12, most NPCs appear to be associated with the inner but not the outer nuclear membrane. Low levels of benzyl alcohol, which increases membrane fluidity, prevented Nup mislocalization and restored the proper localization of Nups that had accumulated in cytoplasmic foci upon a shift to lower temperature. Thus, Apq12p connects nuclear pore biogenesis to the dynamics of the NE.

scholarly journals New Activities of the Nuclear Pore Complexes

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2123
Author(s):  
Richard W. Wong
Keyword(s):  
Small Molecules ◽  
Viral Infections ◽  
Neurological Diseases ◽  
Critical Role ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Special Issue ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Membranes ◽  
Pore Complexes

Nuclear pore complexes (NPCs) at the surface of nuclear membranes play a critical role in regulating the transport of both small molecules and macromolecules between the cell nucleus and cytoplasm via their multilayered spiderweb-like central channel. During mitosis, nuclear envelope breakdown leads to the rapid disintegration of NPCs, allowing some NPC proteins to play crucial roles in the kinetochore structure, spindle bipolarity, and centrosome homeostasis. The aberrant functioning of nucleoporins (Nups) and NPCs has been associated with autoimmune diseases, viral infections, neurological diseases, cardiomyopathies, and cancers, especially leukemia. This Special Issue highlights several new contributions to the understanding of NPC proteostasis.

scholarly journals An ESCRT-LEM protein surveillance system is poised to directly monitor the nuclear envelope and nuclear transport system

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
David J Thaller ◽  
Matteo Allegretti ◽  
Sapan Borah ◽  
Paolo Ronchi ◽  
Martin Beck ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Surveillance System ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Membranes ◽  
Disease Mechanisms ◽  
Pore Complexes ◽  
Membrane Sealing

The integrity of the nuclear membranes coupled to the selective barrier of nuclear pore complexes (NPCs) are essential for the segregation of nucleoplasm and cytoplasm. Mechanical membrane disruption or perturbation to NPC assembly triggers an ESCRT-dependent surveillance system that seals nuclear pores: how these pores are sensed and sealed is ill defined. Using a budding yeast model, we show that the ESCRT Chm7 and the integral inner nuclear membrane (INM) protein Heh1 are spatially segregated by nuclear transport, with Chm7 being actively exported by Xpo1/Crm1. Thus, the exposure of the INM triggers surveillance with Heh1 locally activating Chm7. Sites of Chm7 hyperactivation show fenestrated sheets at the INM and potential membrane delivery at sites of nuclear envelope herniation. Our data suggest that perturbation to the nuclear envelope barrier would lead to local nuclear membrane remodeling to promote membrane sealing. Our findings have implications for disease mechanisms linked to NPC assembly and nuclear envelope integrity.

scholarly journals Actin assembly ruptures the nuclear envelope by prying the lamina away from nuclear pores and nuclear membranes in starfish oocytes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Natalia Wesolowska ◽  
Ivan Avilov ◽  
Pedro Machado ◽  
Celina Geiss ◽  
Hiroshi Kondo ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Super Resolution ◽  
Germinal Vesicle ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Actin Assembly ◽  
Nuclear Membranes ◽  
Pore Complexes ◽  
Distinct Membrane ◽  
Nuclear Rupture

The nucleus of oocytes (germinal vesicle) is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) that are stockpiled for embryonic development. We showed that breakdown of this specialized NE is mediated by an Arp2/3-nucleated F-actin ‘shell’ in starfish oocytes, in contrast to microtubule-driven tearing in mammalian fibroblasts. Here, we address the mechanism of F-actin-driven NE rupture by correlated live-cell, super-resolution and electron microscopy. We show that actin is nucleated within the lamina, sprouting filopodia-like spikes towards the nuclear membranes. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining stretches of membrane accumulate NPCs that are associated with the still-intact lamina. Packed NPCs sort into a distinct membrane network, while breaks appear in ER-like, pore-free regions. We reveal a new function for actin-mediated membrane shaping in nuclear rupture that is likely to have implications in other contexts, such as nuclear rupture observed in cancer cells.

scholarly journals The SUN protein Mps3 controls Ndc1 distribution and function on the nuclear membrane

2014 ◽  
Vol 204 (4) ◽  
pp. 523-539 ◽  
Author(s):  
Jingjing Chen ◽  
Christine J. Smoyer ◽  
Brian D. Slaughter ◽  
Jay R. Unruh ◽  
Sue L. Jaspersen
Keyword(s):  
Spindle Pole Body ◽  
Integral Membrane Protein ◽  
Spindle Pole ◽  
Correlation Spectroscopy ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
The Sun ◽  
Pore Complexes ◽  
And Function

In closed mitotic systems such as Saccharomyces cerevisiae, nuclear pore complexes (NPCs) and the spindle pole body (SPB) must assemble into an intact nuclear envelope (NE). Ndc1 is a highly conserved integral membrane protein involved in insertion of both complexes. In this study, we show that Ndc1 interacts with the SUN domain–containing protein Mps3 on the NE in live yeast cells using fluorescence cross-correlation spectroscopy. Genetic and molecular analysis of a series of new ndc1 alleles allowed us to understand the role of Ndc1–Mps3 binding at the NE. We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication. Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component. Based on our data we propose that the Ndc1–Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB.

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