scholarly journals Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis

2013 ◽  
Vol 203 (6) ◽  
pp. 883-893 ◽  
Author(s):  
Nina Schweizer ◽  
Cristina Ferrás ◽  
David M. Kern ◽  
Elsa Logarinho ◽  
Iain M. Cheeseman ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Half Life ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Limiting Factor ◽  
Nuclear Pore ◽  
Independent Manner ◽  
Unknown Mechanism ◽  
Rate Limiting

Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1–c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1–c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response.

scholarly journals The yeast nuclear pore complex functionally interacts with components of the spindle assembly checkpoint

2002 ◽  
Vol 159 (5) ◽  
pp. 807-819 ◽  
Author(s):  
Tatiana Iouk ◽  
Oliver Kerscher ◽  
Robert J. Scott ◽  
Munira A. Basrai ◽  
Richard W. Wozniak
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Genetic Analysis ◽  
Nuclear Pore Complex ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Nuclear Pore ◽  
Yeast Saccharomyces Cerevisiae ◽  
Functional Link

Aphysical and functional link between the nuclear pore complex (NPC) and the spindle checkpoint machinery has been established in the yeast Saccharomyces cerevisiae. We show that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle. There they are associated with a subcomplex of nucleoporins containing Nup53p, Nup170p, and Nup157p. The association of the Mad1p–Mad2p complex with the NPC requires Mad1p and is mediated in part by Nup53p. On activation of the spindle checkpoint, we detect changes in the interactions between these proteins, including the release of Mad2p (but not Mad1p) from the NPC and the accumulation of Mad2p at kinetochores. Accompanying these events is the Nup53p-dependent hyperphosphorylation of Mad1p. On the basis of these results and genetic analysis of double mutants, we propose a model in which Mad1p bound to a Nup53p-containing complex sequesters Mad2p at the NPC until its release by activation of the spindle checkpoint. Furthermore, we show that the association of Mad1p with the NPC is not passive and that it plays a role in nuclear transport.

scholarly journals Ran GTPase Regulates Mad2 Localization to the Nuclear Pore Complex

2005 ◽  
Vol 4 (2) ◽  
pp. 274-280 ◽  
Author(s):  
B. Booth Quimby ◽  
Alexei Arnaoutov ◽  
Mary Dasso
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Spindle Assembly Checkpoint ◽  
Nuclear Export Signal ◽  
Spindle Assembly ◽  
Nuclear Pore ◽  
Ran Gtpase ◽  
Nuclear Levels ◽  
Nucleocytoplasmic Distribution

ABSTRACT In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the nuclear pore complex (NPC) during interphase. Deletion of MAD1 or MAD2 did not affect steady-state nucleocytoplasmic distribution of a classical nuclear localization signal-containing reporter, a nuclear export signal-containing reporter, or Ran localization. We utilized cells with conditional mutations in the yeast Ran GTPase pathway to examine the relationship between Ran and targeting of checkpoint regulators to the NPC. Mutations that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. Our observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.

scholarly journals The Torsin/ NEP1R1-CTDNEP1/ Lipin axis regulates nuclear envelope lipid metabolism for nuclear pore complex insertion

2020 ◽  
Author(s):  
Julie Jacquemyn ◽  
Joyce Foroozandeh ◽  
Katlijn Vints ◽  
Jef Swerts ◽  
Patrik Verstreken ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Lipid Droplets ◽  
Nuclear Pore ◽  
List Type ◽  
Unknown Mechanism ◽  
Structure Lipid ◽  
Cellular Events ◽  
Upstream Regulator

AbstractTorsin ATPases of the endoplasmic reticulum (ER) and nuclear envelope (NE) lumen inhibit Lipin-mediated phosphatidate (PA) to diacylglycerol (DAG) conversion by an unknown mechanism. This excess PA metabolism is implicated in TOR1A/TorsinA diseases, but it is unclear whether it explains why Torsin concomitantly affects nuclear structure, lipid droplets (LD), organelle and cell growth. Here a fly miniscreen identified that Torsins affect these events via the NEP1R1-CTDNEP1 phosphatase complex. Further, Torsin homo-oligomerization rather than ATPase activity was key to function. NEP1R1-CTDNEP1 activates Lipin by dephosphorylation. We show that Torsin prevents CTDNEP1 from accumulating in the NE and excludes Lipin from the nucleus. Moreover, this repression of nuclear PA metabolism is required for interphase nuclear pore biogenesis. We conclude that Torsin is an upstream regulator of the NEP1R1-CTDNEP1/ Lipin pathway. This connects the ER/NE lumen with PA metabolism, and affects numerous cellular events including it has a previously unrecognized role in nuclear pore biogenesis.HighlightsNuclear envelope PA-DAG-TAG synthesis is independently regulated by Torsin and Torip/LAP1Torsin removes CTDNEP1 from the nuclear envelope and excludes Lipin from the nucleusExcess nuclear envelope NEP1R1-CTDNEP1/ Lipin activity impairs multiple aspects of NPC biogenesisNEP1R1-CTDNEP1/ Lipin inhibition prevents cellular defects associated with TOR1A and TOR1AIP1 / LAP1 disease

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 An agent-based model for mRNA export through the nuclear pore complex

2014 ◽  
Vol 25 (22) ◽  
pp. 3643-3653 ◽  
Author(s):  
Mohammad Azimi ◽  
Evgeny Bulat ◽  
Karsten Weis ◽  
Mohammad R. K. Mofrad
Keyword(s):  
Nuclear Pore Complex ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Central Channel ◽  
Agent Based Model ◽  
Agent Based ◽  
Transport Dynamics ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics.

scholarly journals Binding Dynamics of Structural Nucleoporins Govern Nuclear Pore Complex Permeability and May Mediate Channel Gating

2003 ◽  
Vol 23 (2) ◽  
pp. 534-542 ◽  
Author(s):  
Nataliya Shulga ◽  
David S. Goldfarb
Keyword(s):  
Nuclear Pore Complex ◽  
Size Range ◽  
Channel Gating ◽  
Aliphatic Alcohols ◽  
Complex Permeability ◽  
Nuclear Pore ◽  
Wild Type ◽  
Unknown Mechanism ◽  
Reversible Dissociation ◽  
Wide Size Range

ABSTRACT The nuclear pore complex (NPC) is a permeable sieve that can dilate to facilitate the bidirectional translocation of a wide size range of receptor-cargo complexes. The binding of receptors to FG nucleoporin docking sites triggers channel gating by an unknown mechanism. Previously, we used deoxyglucose and chilling treatments to implicate Nup170p and Nup188p in the control of NPC sieving in Saccharomyces cerevisiae. Here, we report that aliphatic alcohols increase the permeability of wild-type and nup170Δ NPCs. In conjunction with increases in permeability, aliphatic alcohols, deoxyglucose, and chilling trigger the reversible dissociation of several nucleoporins from nup170Δ NPCs. These results are consistent with the hypothesis that NPC gating occurs when molecular latches composed of FG repeats and structural nucleoporins dissociate.

scholarly journals Dissection of the NUP107 nuclear pore subcomplex reveals a novel interaction with spindle assembly checkpoint protein MAD1 in Caenorhabditis elegans

2012 ◽  
Vol 23 (5) ◽  
pp. 930-944 ◽  
Author(s):  
Eduardo Ródenas ◽  
Cristina González-Aguilera ◽  
Cristina Ayuso ◽  
Peter Askjaer
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Envelope ◽  
Molecular Mechanisms ◽  
Spindle Assembly Checkpoint ◽  
Protein Import ◽  
Spindle Assembly ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Pore Complexes

Nuclear pore complexes consist of several subcomplexes. The NUP107 complex is important for nucleocytoplasmic transport, nuclear envelope assembly, and kinetochore function. However, the underlying molecular mechanisms and the roles of individual complex members remain elusive. We report the first description of a genetic disruption of NUP107 in a metazoan. Caenorhabditis elegans NUP107/npp-5 mutants display temperature-dependent lethality. Surprisingly, NPP-5 is dispensable for incorporation of most nucleoporins into nuclear pores and for nuclear protein import. In contrast, NPP-5 is essential for proper kinetochore localization of NUP133/NPP-15, another NUP107 complex member, whereas recruitment of NUP96/NPP-10C and ELYS/MEL-28 is NPP-5 independent. We found that kinetochore protein NUF2/HIM-10 and Aurora B/AIR-2 kinase are less abundant on mitotic chromatin upon NPP-5 depletion. npp-5 mutants are hypersensitive to anoxia, suggesting that the spindle assembly checkpoint (SAC) is compromised. Indeed, NPP-5 interacts genetically and physically with SAC protein MAD1/MDF-1, whose nuclear envelope accumulation requires NPP-5. Thus our results strengthen the emerging connection between nuclear pore proteins and chromosome segregation.

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