Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration

The nuclear import receptors importin β and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin β, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107–160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin β and transportin “global positioning system”or “GPS” pathways that are mechanistically parallel.

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The yeast nucleoporin Nsp1 binds nuclear localization sequences in vitro

Biochemistry and Cell Biology ◽

10.1139/o96-039 ◽

1996 ◽

Vol 74 (3) ◽

pp. 363-372 ◽

Cited By ~ 9

Author(s):

Werner Barth ◽

Ursula Stochaj

Keyword(s):

Nuclear Localization ◽

Specific Binding ◽

T Antigen ◽

Nuclear Localization Sequence ◽

Nuclear Pore ◽

Sv40 T Antigen ◽

Nuclear Targeting ◽

Nuclear Localization Sequences

Facilitated transport of proteins into the nucleus requires nuclear localization sequences (NLSs) be present in the protein destined for the nucleus. The specific binding of NLSs by components of the nuclear transport apparatus is essential for these targeting reactions. We now report that the yeast nucleoporin Nsp1 binds specifically nuclear localization sequences in vitro. This nucleoporin recognizes several NLSs that are functional for nuclear targeting in vivo, including the NLS of SV40 T-antigen and of the yeast transcription factor Gal4. Nsp1 is organized into three domains, and we have located NLS binding sites to the N-terminal portion and the middle repetitive region of the protein. For the interaction between the NLS of SV40 T-antigen and Nsp1, we obtained association constants of 1.2 × 107 M−1 and 5 × 107 M−1. An association constant of 5 × 107 M−1 was determined for NLS binding to the repetitive domain of Nsp1. We analyzed binding of Nsp1 and its domains to a mutant version of the NLS derived from SV40 T-antigen, which poorly functions for nuclear targeting in vivo. The affinity for the mutant signal was about two orders of magnitude lower than for the wild-type NLS.Key words: Nsp1, nuclear pore complex, nucleoporin, nuclear localization sequence, protein targeting, yeast.

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The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

The Journal of Cell Biology ◽

10.1083/jcb.200202088 ◽

2002 ◽

Vol 158 (1) ◽

pp. 63-77 ◽

Cited By ~ 140

Author(s):

Tobias C. Walther ◽

Helen S. Pickersgill ◽

Volker C. Cordes ◽

Martin W. Goldberg ◽

Terry D. Allen ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Protein Import ◽

Nuclear Localization Sequence ◽

Nuclear Pore ◽

Slight Reduction ◽

Cytoplasmic Filaments ◽

Localization Sequence ◽

Importin Α

The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin α/β– or transportin-dependent import.

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Nup62 is recruited to pathological condensates and promotes TDP-43 insolubility in C9orf72 and sporadic ALS/FTLD.

10.21203/rs.3.rs-144654/v1 ◽

2021 ◽

Author(s):

Amanda Gleixner ◽

Brandie Morris Verdone ◽

Charlton Otte ◽

Nandini Ramesh ◽

Jenna Gale ◽

...

Keyword(s):

Phase Transitions ◽

Nucleocytoplasmic Transport ◽

Nuclear Localization Sequence ◽

Nuclear Pore ◽

Permeability Barrier ◽

Nucleocytoplasmic Trafficking ◽

Mechanisms Of Toxicity ◽

Sporadic Als

Abstract Amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) share clinical, neuropathological, and genetic features. This includes common genetic disease-causing mutations such as the expanded G4C2 repeat in the C9orf72 gene (C9-ALS/FTLD) and cytoplasmic and insoluble protein depositions of the TDP-43 in degenerating regions of the brain and spinal cord. Proposed mechanisms of toxicity in C9-ALS/FTLD are the production of repeat expansion transcripts and their dipeptide repeat proteins (DPRs) products which are hypothesized to drive nucleocytoplasmic transport defects. The nuclear pore complex (NPC) regulates nucleocytoplasmic trafficking by creating a selectivity and permeability barrier comprised of phenylalanine glycine nucleoporins (FG nups). However, the relationship between FG nups and TDP-43 pathology remains elusive. Here, we define two mechanisms through which TDP-43 promotes Nup62 nuclear depletion and cytoplasmic in C9-ALS/FTLD and sALS/FTLD. In C9-ALS/FTLD, poly-GR initiates the formation of TDP-43 containing stress granules (SGs) that trigger the nuclear loss and recruitment of Nup62 in vitro and in vivo. When colocalized, cytoplasmic TDP-43:Nup62 assemblies mature into insoluble inclusions through an interaction within the TDP-43 nuclear localization sequence (NLS) suggesting Nup62 promotes deleterious phase transitions. Absent of poly-GR, aberrant TDP-43 phase transitions in the cytoplasm recruits and mislocalizes Nup62 into pathological inclusions. The result of these cytoplasmic Nup62 and TDP-43 interactions are pathological and insoluble TDP-43:Nup62 assemblies that are observed in C9-ALS/FTLD and sALS/FTLD CNS tissue.

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The 2μm Plasmid Stability System: Analyses of the Interactions among Plasmid- and Host-Encoded Components

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7466 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7466-7477 ◽

Cited By ~ 29

Author(s):

Soundarapandian Velmurugan ◽

Yong-Tae Ahn ◽

Xian-Mei Yang ◽

Xu-Li Wu ◽

Makkuni Jayaram

Keyword(s):

Plasmid Stability ◽

High Specificity ◽

Nuclear Localization Sequence ◽

Nuclear Targeting ◽

Amino Terminal ◽

Rep Proteins ◽

Localization Sequence ◽

Carboxy Terminal

ABSTRACT The stable inheritance of the 2μm plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with thecis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2μm circle-derived plasmid shows relatively poor stability.

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Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13

Molecular and Cellular Biology ◽

10.1128/mcb.24.11.4824-4834.2004 ◽

2004 ◽

Vol 24 (11) ◽

pp. 4824-4834 ◽

Cited By ~ 34

Author(s):

Jonathan E. Ploski ◽

Monee K. Shamsher ◽

Aurelian Radu

Keyword(s):

Transcription Factor ◽

In Vitro Assays ◽

Nuclear Localization Sequence ◽

Amino Acid Residues ◽

Homeodomain Transcription Factor ◽

Permeabilized Cells ◽

Localization Sequence ◽

Direct Binding

ABSTRACT We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin β family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13.

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Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export

Molecular and Cellular Biology ◽

10.1128/mcb.20.15.5619-5630.2000 ◽

2000 ◽

Vol 20 (15) ◽

pp. 5619-5630 ◽

Cited By ~ 83

Author(s):

Tinglu Guan ◽

Ralph H. Kehlenbach ◽

Eric C. Schirmer ◽

Angelika Kehlenbach ◽

Fan Fan ◽

...

Keyword(s):

Rat Liver ◽

Nuclear Export ◽

Nuclear Protein ◽

Protein Export ◽

Immunogold Electron Microscopy ◽

Nuclear Localization Sequence ◽

Nuclear Pore ◽

Direct Role ◽

Localization Sequence

ABSTRACT We present here a detailed analysis of a rat polypeptide termed Nup50 (formerly NPAP60) that was previously found to be associated with the nuclear pore complex (F. Fan et al., Genomics 40:444–453, 1997). We have found that Nup50 (and/or a related 70-kDa polypeptide) is present in numerous rat cells and tissues. By immunofluorescence microscopy, Nup50 was found to be highly concentrated at the nuclear envelope of rat liver nuclei, whereas in cultured NRK cells it also is abundant in intranuclear regions. On the basis of immunogold electron microscopy of both rat liver nuclear envelopes and NRK cells, we determined that Nup50 is specifically localized in the nucleoplasmic fibrils of the pore complex. Microinjection of anti-Nup50 antibodies into the nucleus of NRK cells resulted in strong inhibition of nuclear export of a protein containing a leucine-rich nuclear export sequence, whereas nuclear import of a protein containing a classical nuclear localization sequence was unaffected. Correspondingly, CRM1, the export receptor for leucine-rich export sequences, directly bound to a fragment of Nup50 in vitro, whereas several other import and export receptors did not significantly interact with this fragment. Taken together, our data indicate that Nup50 has a direct role in nuclear protein export and probably serves as a binding site on the nuclear side of the pore complex for export receptor-cargo complexes.

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A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-β and the viral helicase, NS3

Journal of General Virology ◽

10.1099/0022-1317-82-4-735 ◽

2001 ◽

Vol 82 (4) ◽

pp. 735-745 ◽

Cited By ~ 116

Author(s):

Magnus Johansson ◽

Andrew J. Brooks ◽

David A. Jans ◽

Subhash G. Vasudevan

Keyword(s):

Dengue Virus ◽

Rna Polymerase ◽

Direct Interaction ◽

Small Region ◽

Terminal Region ◽

Nuclear Localization Sequence ◽

Rna Dependent Rna Polymerase ◽

Localization Sequence

The dengue virus RNA-dependent RNA polymerase, NS5, and the protease/helicase, NS3, are multidomain proteins that have been shown to interact both in vivo and in vitro. A hyperphosphorylated form of NS5 that does not interact with NS3 has been detected in the nuclei of virus-infected cells, presumably as the result of the action of a functional nuclear localization sequence within the interdomain region of NS5 (residues 369–405). In this study, it is shown by using the yeast two-hybrid system that the C-terminal region of NS3 (residues 303–618) interacts with the N-terminal region of NS5 (residues 320–368). Further, it is shown that this same region of NS5 is also recognized by the cellular nuclear import receptor importin-β. The interaction between NS5 and importin-β and competition by NS3 with the latter for the same binding site on NS5 were confirmed by pull-down assays. The direct interaction of importin-β with NS5 has implications for the mechanism by which this normally cytoplasmic protein may be targetted to the nucleus.

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TNP-470 Suppresses the Tumorigenicity of HT1080 Fibrosarcoma Tumor Through the Inhibition of VEGF Secretion From the Tumor Cells

Sarcoma ◽

10.1080/13577140120099182 ◽

2001 ◽

Vol 5 (4) ◽

pp. 197-202 ◽

Cited By ~ 6

Author(s):

Mitsunori Kaya ◽

Takuro Wada ◽

Satoshi Nagoya ◽

Satoshi Kawaguchi ◽

Toshihiko Yamashita ◽

...

Keyword(s):

Conditioned Medium ◽

Direct Action ◽

Angiogenesis Inhibitor ◽

Angiogenesis Inhibitors ◽

Fibrosarcoma Cells ◽

Ht1080 Cells ◽

Human Fibrosarcoma Cells ◽

And Migration

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer. TNP-470, a systemic analogue of fumagillin, is an angiogenesis inhibitor capable of suppressing the tumorigenicity in several animal models even though the mechanisms of action have not been completely clarified. In the current study, we investigated the effects of TNP-470 on human fibrosarcoma cellsin vivoandin vitro. The administration of TNP-470 could suppress the tumorigenicity of HT1080 fibrosarcoma tumor. The conditioned medium from HT1080 fibrosarcoma cells treated with TNP-470 inhibited the proliferation and migration of human endothelial cell line, HUVEC and ECV304. The concentration of VEGF in the conditioned medium from HT1080 cells treated with TNP-470 was lower than that of the cells without TNP-470 treatment, indicating that TNP-470 downregulates the secretion of VEGF from HT1080 cells. These findings strongly suggest that the direct action of TNP-470 on sarcoma cells inhibits angiogenesis through the downregulation of VEGF secretion and this angiogenesis suppression resulted in the inhibition of tumorigenicity of HT1080 fibrosarcoma tumo.

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Mechanism of phosphaturia elicited by administration of phosphonoformate in vivo

AJP Renal Physiology ◽

10.1152/ajprenal.1988.255.5.f984 ◽

1988 ◽

Vol 255 (5) ◽

pp. F984-F994 ◽

Cited By ~ 5

Author(s):

M. VanScoy ◽

M. Loghman-Adham ◽

M. Onsgard ◽

M. Szczepanska-Konkel ◽

S. Homma ◽

...

Keyword(s):

Direct Action ◽

Fractional Excretion ◽

Proximal Tubules ◽

Camp Phosphodiesterase ◽

Luminal Side ◽

Metabolic Transformation ◽

Rat Renal Cortical Slices ◽

Fractional Excretion Of Phosphate

We examined whether phosphonoformate (PFA) can cause phosphaturia through its direct action on brush-border membrane (BBM) in vivo. Infusion of PFA or of parathyroid hormone (PTH) to thyroparathyroidectomized rats caused a marked increase in fractional excretion of phosphate without changes in excretion of Na+ or of GFR. The PFA-induced phosphaturia was not accompanied by an increase in urinary adenosine-3',5'-cyclic monophosphate (cAMP); moreover, PFA added in vitro did not influence the PTH-sensitive adenylate cyclase and cAMP-phosphodiesterase in proximal convoluted tubules. In BBM vesicles (BBMV) from rats with PFA-elicited phosphaturia, neither the rate of Na+-Pi symport nor Na+-dependent binding of [14C]PFA on BBMV was changed, whereas in BBMV from PTH-infused rats the Vmax of Na+-Pi symport decreased. PFA is almost completely ultrafiltrable; no metabolic transformation of PFA was detected after [14C]PFA exposure to rat renal cortical slices, homogenate, or to blood. We conclude that PFA causes phosphaturia by direct inhibition of Na+-Pi symport across BBM in proximal tubules, acting from the luminal side. Thus PFA (foscarnet) has a unique direct mechanism of phosphaturic effect, via its action on Pi reabsorption in proximal tubules in vivo.

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Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽

10.7554/elife.10785 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 44

Author(s):

Andrei Vovk ◽

Chad Gu ◽

Michael G Opferman ◽

Larisa E Kapinos ◽

Roderick YH Lim ◽

...

Keyword(s):

Spatial Organization ◽

Intrinsically Disordered Proteins ◽

Nucleocytoplasmic Transport ◽

Transport Proteins ◽

Disordered Proteins ◽

Biophysical Model ◽

Nuclear Pore ◽

Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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