scholarly journals Interactions between a Nuclear Transporter and a Subset of Nuclear Pore Complex Proteins Depend on Ran GTPase

1999 ◽  
Vol 19 (2) ◽  
pp. 1547-1557 ◽  
Author(s):  
Matthias Seedorf ◽  
Marc Damelin ◽  
Jason Kahana ◽  
Tetsuya Taura ◽  
Pamela A. Silver
Keyword(s):  
Nuclear Pore Complex ◽  
Fluorescent Protein ◽  
Bound State ◽  
Yeast Cells ◽  
Pore Channel ◽  
Nuclear Pore ◽  
Mutant Form ◽  
Dependent Manner ◽  
Functional Protein ◽  
Ran Gtpase

ABSTRACT Proteins to be transported into the nucleus are recognized by members of the importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released, the importin then moves back through the channel for new rounds of transport. Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detail. Pse1-GFP is in a complex with importin-α and -β (Srp1p and Kap95p in yeast cells) that is sensitive to the nucleotide-bound state of the Ran GTPase. In addition, Pse1p associates with the nucleoporins Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p show different patterns of interaction with nucleoporins. Association of Pse1p with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound state, the nucleoporin association is lost. A mutant form of Pse1p that does not bind Ran also fails to interact with nucleoporins. These data indicate that transport receptors such as Pse1p interact in a Ran-dependent manner with certain nucleoporins. These nucleoporins may represent major docking sites for Pse1p as it moves in or out of the nucleus via the NPC.

scholarly journals Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

2018 ◽  
Vol 115 (17) ◽  
pp. E3969-E3977 ◽  
Author(s):  
Sasikumar Rajoo ◽  
Pascal Vallotton ◽  
Evgeny Onischenko ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Altered Expression ◽  
Copy Numbers ◽  
Quantitative Image ◽  
Multiple Copies ◽  
Almost All ◽  
High Degree

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

scholarly journals The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Alyssa N. Coyne ◽  
Jeffrey D. Rothstein
Keyword(s):  
Nuclear Pore Complex ◽  
Mammalian Cells ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Structured Illumination Microscopy ◽  
Neuronal Nuclei ◽  
Complex Injury ◽  
Sporadic Als ◽  
Human Neurons

AbstractNuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described.

scholarly journals Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

2009 ◽  
Vol 185 (3) ◽  
pp. 475-491 ◽  
Author(s):  
Evgeny Onischenko ◽  
Leslie H. Stanton ◽  
Alexis S. Madrid ◽  
Thomas Kieselbach ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Interaction Network ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Binding Partners ◽  
Interaction Partners ◽  
Redundant Functions

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

scholarly journals The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

2001 ◽  
Vol 152 (2) ◽  
pp. 385-400 ◽  
Author(s):  
Patrick Heun ◽  
Thierry Laroche ◽  
M.K. Raghuraman ◽  
Susan M. Gasser
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Yeast Cells ◽  
G1 Phase ◽  
Nuclear Pore ◽  
Origin Firing ◽  
Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

scholarly journals Simple kinetic relationships and nonspecific competition govern nuclear import rates in vivo

2006 ◽  
Vol 175 (4) ◽  
pp. 579-593 ◽  
Author(s):  
Benjamin L. Timney ◽  
Jaclyn Tetenbaum-Novatt ◽  
Diana S. Agate ◽  
Rosemary Williams ◽  
Wenzhu Zhang ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Yeast Cells ◽  
Limiting Factor ◽  
Nuclear Pore ◽  
Nuclear Localization Signals ◽  
The Poor ◽  
Cytoplasmic Proteins

Many cargoes destined for nuclear import carry nuclear localization signals that are recognized by karyopherins (Kaps). We present methods to quantitate import rates and measure Kap and cargo concentrations in single yeast cells in vivo, providing new insights into import kinetics. By systematically manipulating the amounts, types, and affinities of Kaps and cargos, we show that import rates in vivo are simply governed by the concentrations of Kaps and their cargo and the affinity between them. These rates fit to a straightforward pump–leak model for the import process. Unexpectedly, we deduced that the main limiting factor for import is the poor ability of Kaps and cargos to find each other in the cytoplasm in a background of overwhelming nonspecific competition, rather than other more obvious candidates such as the nuclear pore complex and Ran. It is likely that most of every import round is taken up by Kaps and nuclear localization signals sampling other cytoplasmic proteins as they locate each other in the cytoplasm.

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.

scholarly journals Author response: Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner

2015 ◽  
Author(s):  
Alan R Lowe ◽  
Jeffrey H Tang ◽  
Jaime Yassif ◽  
Michael Graf ◽  
William YC Huang ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Author Response ◽  
Nuclear Pore ◽  
Dependent Manner

scholarly journals Dynamics of Nuclear Pore Distribution in Nucleoporin Mutant Yeast Cells

1997 ◽  
Vol 136 (4) ◽  
pp. 747-759 ◽  
Author(s):  
Naïma Belgareh ◽  
Valérie Doye
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Pore Distribution ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Dividing Cells ◽  
Green Fluorescent ◽  
Amino Terminal Domain

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133− dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p- induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133− mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.

scholarly journals Karyopherins regulate nuclear pore complex barrier and transport function

2017 ◽  
Vol 216 (11) ◽  
pp. 3609-3624 ◽  
Author(s):  
Larisa E. Kapinos ◽  
Binlu Huang ◽  
Chantal Rencurel ◽  
Roderick Y.H. Lim
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Nuclear Localization Signal ◽  
Barrier Function ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Transport Function ◽  
Guanosine Triphosphate ◽  
Localization Signal

Nucleocytoplasmic transport is sustained by karyopherins (Kaps) and a Ran guanosine triphosphate (RanGTP) gradient that imports nuclear localization signal (NLS)–specific cargoes (NLS-cargoes) into the nucleus. However, how nuclear pore complex (NPC) barrier selectivity, Kap traffic, and NLS-cargo release are systematically linked and simultaneously regulated remains incoherent. In this study, we show that Kapα facilitates Kapβ1 turnover and occupancy at the NPC in a RanGTP-dependent manner that is directly coupled to NLS-cargo release and NPC barrier function. This is underpinned by the binding affinity of Kapβ1 to phenylalanine–glycine nucleoporins (FG Nups), which is comparable with RanGTP·Kapβ1, but stronger for Kapα·Kapβ1. On this basis, RanGTP is ineffective at releasing standalone Kapβ1 from NPCs. Depleting Kapα·Kapβ1 by RanGTP further abrogates NPC barrier function, whereas adding back Kapβ1 rescues it while Kapβ1 turnover softens it. Therefore, the FG Nups are necessary but insufficient for NPC barrier function. We conclude that Kaps constitute integral constituents of the NPC whose barrier, transport, and cargo release functionalities establish a continuum under a mechanism of Kap-centric control.

scholarly journals Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity

2014 ◽  
Vol 25 (9) ◽  
pp. 1421-1436 ◽  
Author(s):  
Jennifer M. Holden ◽  
Ludek Koreny ◽  
Samson Obado ◽  
Alexander V. Ratushny ◽  
Wei-Ming Chen ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Mitotic Spindle ◽  
Nuclear Periphery ◽  
Coiled Coil ◽  
Nuclear Lamina ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Major Barrier ◽  
African Trypanosomes

The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle–dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina.

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