Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum

The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions.

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The Formation, Structure, Distribution and Frequency of Nuclear Pores

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066644 ◽

1971 ◽

Vol 29 ◽

pp. 518-519

Author(s):

G. G. Maul

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Membrane ◽

Dynamic Structure ◽

Nuclear Pore ◽

Nuclear Pores ◽

Filter Function ◽

Etching Technique ◽

Selective Filter ◽

Membranous Part

The chromatin of eukaryotic cells is separated from the cytoplasm by a double membrane. One obvious structural specialization of the nuclear membrane is the presence of pores which have been implicated to facilitate the selective nucleocytoplasmic exchange of a variety of large molecules. Thus, the function of nuclear pores has mainly been regarded to be a passive one. Non-membranous diaphragms, radiating fibers, central rings, and other pore-associated structures were thought to play a role in the selective filter function of the nuclear pore complex. Evidence will be presented that suggests that the nuclear pore is a dynamic structure which is non-randomly distributed and can be formed during interphase, and that a close relationship exists between chromatin and the membranous part of the nuclear pore complex.Octagonality of the nuclear pore complex has been confirmed by a variety of techniques. Using the freeze-etching technique, it was possible to show that the membranous part of the pore complex has an eight-sided outline in human melanoma cells in vitro. Fibers which traverse the pore proper at its corners are continuous and indistinguishable from chromatin at the nucleoplasmic side, as seen in conventionally fixed and sectioned material. Chromatin can be seen in octagonal outline if serial sections are analyzed which are parallel but do not include nuclear membranes (Fig. 1). It is concluded that the shape of the pore rim is due to fibrous material traversing the pore, and may not have any functional significance. In many pores one can recognize a central ring with eight fibers radiating to the corners of the pore rim. Such a structural arrangement is also found to connect eight ribosomes at the nuclear membrane.

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

Biological Chemistry ◽

10.1515/hsz-2013-0285 ◽

2014 ◽

Vol 395 (5) ◽

pp. 515-528 ◽

Cited By ~ 42

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.

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Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

10.1101/2019.12.16.878546 ◽

2019 ◽

Author(s):

Cassandra K. Hayne ◽

Casey A. Schmidt ◽

A. Gregory Matera ◽

Robin E. Stanley

Keyword(s):

Animal Cells ◽

Trna Splicing ◽

Polynucleotide Kinase ◽

Genes Encoding ◽

Intron Removal ◽

And Function ◽

Insight Into ◽

Negative Modulator

ABSTRACTThe splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

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

10.1101/2020.07.05.188599 ◽

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

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Secretion ofSaccharomyces cerevisiaeKiller Toxin: Processing of the Glycosylated Precursor

Molecular and Cellular Biology ◽

10.1128/mcb.3.8.1362 ◽

1983 ◽

Vol 3 (8) ◽

pp. 1362-1370 ◽

Cited By ~ 61

Author(s):

H. Bussey ◽

D. Saville ◽

D. Greene ◽

D. J. Tipper ◽

K. A. Bostian

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Killer Toxin ◽

Membrane System ◽

Restrictive Temperature ◽

Wild Type ◽

Endoproteolytic Cleavage ◽

Toxin Secretion

Killer toxin secretion was blocked at the restrictive temperature inSaccharomyces cerevisiae secmutants with conditional defects in theS. cerevisiaesecretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in allsecmutants at the restrictive temperature. Insec18the protoxin was stable after a chase; but insec7andsec1the protoxin was unstable, and insec111K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and insec7andsec1cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined twokexmutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable inkex1but unstable inkex2. Protoxin stability inkex1 kex2double mutants indicated the orderkex1→kex2in the protoxin processing pathway. TPCK did not block protoxin instability inkex2mutants. This suggested that theKEX1- andKEX2-dependent steps preceded thesec7Golgi block. We attempted to localize the protoxin inS. cerevisiaecells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.

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Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p, Nup59p, and Nup170p

The Journal of Cell Biology ◽

10.1083/jcb.143.7.1813 ◽

1998 ◽

Vol 143 (7) ◽

pp. 1813-1830 ◽

Cited By ~ 120

Author(s):

Marcello Marelli ◽

John D. Aitchison ◽

Richard W. Wozniak

Keyword(s):

Nuclear Pore Complex ◽

Core Protein ◽

Specific Binding ◽

Protein A ◽

Small Gtpase ◽

Nuclear Pore ◽

Binding Assays ◽

Reporter Protein ◽

Physiological Relevance

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p–protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only β-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.

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Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

Nucleic Acids Research ◽

10.1093/nar/gkaa438 ◽

2020 ◽

Vol 48 (14) ◽

pp. 7609-7622 ◽

Cited By ~ 3

Author(s):

Cassandra K Hayne ◽

Casey A Schmidt ◽

Maira I Haque ◽

A Gregory Matera ◽

Robin E Stanley

Keyword(s):

Animal Cells ◽

Trna Splicing ◽

Polynucleotide Kinase ◽

Genes Encoding ◽

Intron Removal ◽

And Function ◽

Insight Into ◽

Negative Modulator

Abstract The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

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Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran

Molecular Biology of the Cell ◽

10.1091/mbc.11.2.703 ◽

2000 ◽

Vol 11 (2) ◽

pp. 703-719 ◽

Cited By ~ 26

Author(s):

Susanne M. Steggerda ◽

Ben E. Black ◽

Bryce M. Paschal

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Protein Import ◽

Nuclear Translocation ◽

Living Cells ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Permeabilized Cells ◽

Dependent Protein

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

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From Signalosome to Enhanceosome: The Translocation of CD40 from Membrane to Nucleus Involves Multiple Mechanisms

Blood ◽

10.1182/blood.v112.11.3781.3781 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3781-3781

Author(s):

Hai-Jun Zhou ◽

Archie Tamayo ◽

Lan Pham ◽

Yen-Chiu Lin-Lee ◽

Richard J. Ford

Keyword(s):

Endoplasmic Reticulum ◽

B Cell ◽

B Lymphocytes ◽

Nuclear Localization ◽

Cell Nucleus ◽

Nuclear Pore Complex ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Nuclear Pore ◽

Lymphoma Cells

Abstract CD40 plays important roles in the proliferation, survival and differentiation of lymphocytes. Constitutively active CD40 recruits TRAFs and IKKs within the lipid rafts to form a signalosome that mediates pivotal downstream proliferation and survival mechanisms involving NF-kB. Recently, we have reported that nuclear localization of CD40, through its interaction with c-Rel, promotes growth, cell cycle progression and survival in large B cell lymphoma. Our studies have opened a new paradigm in the functional role of CD40 in non-Hodgkin lymphomas of B cell origin (NHL-B). However, the mechanism about how CD40 enters nuclear still remains elusive. In this study, we show that CD40 ligation enhances its nuclear accumulation with activation of c-Rel in both normal B-lymphocytes and B cell lymphoma cells with cell fractionation assay and con-focal microscopy. Over-expression of c-Rel in B cell lymphoma cells drives CD40 into cell nucleus. We hypothesize that the route CD40 enters nucleus may involve endosome-endoplasmic reticulum-nuclear pore complex. Indeed, further studies show CD40 co-localizes with endosome marker-EEA1 and endoplasmic reticulum marker-Sec61. Furthermore, our co-immunoprecipitation assay has demonstrated CD40 interacts with Sec61. CD40 also co-localizes and immuno-precipitates with nuclear pore complex (NPC) proteins-NUP62 in normal B-lymphocytes and B lymphoma cells, which suggests NPC proteins may facilitate the nuclear translocation of CD40 protein. Overall, our study suggests that translocation of CD40 into cell nucleus involves multiple pathways. Blocking nuclear localization may modulate the function of CD40 in lymphoma cells; which could provide a new-targeted therapeutic approach for lymphoma therapy.

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