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The Essential Yeast Nucleoporin NUP159 Is Located on the Cytoplasmic Side of the Nuclear Pore Complex and Serves in Karyopherin-mediated Binding of Transport Substrate

Authors:
Doris M. Kraemer
Doris M. Kraemer
Doris M. Kraemer
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Caterina Strambio De Castillia at University of Massachusetts Medical School
Caterina Strambio De Castillia
Günter Blobel
Günter Blobel
Günter Blobel
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Michael P Rout at The Rockefeller University
Michael P Rout
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Abstract

We have identified a new yeast nucleoporin of 159 kDa that we term NUP159. Immunofluorescence microscopy with a monospecific monoclonal antibody against NUP159 gave the punctate nuclear rim staining characteristic of nucleoporins. Immunogold electron microscopy with isolated yeast NEs yielded decoration of only the cytoplasmic side of the nuclear pore complex. The gene encoding NUP159 is essential, and, like some other nucleoporins, NUP159 contains a coiled-coil domain as well as a domain of repeated motifs. Five segments of NUP159, covering its entire length, were expressed in Escherichia coli. The repeat motif-containing segment was found to bind a nuclear transport substrate in the presence of vertebrate cytosolic extract containing nuclear transport factors. This segment also bound 35S-labeled mammalian karyopherin beta, one such transport factor that mediates the docking of substrates to the nuclear pore complex. These data establish a direct biochemical link between the repeat motif domain of a yeast nucleoporin, transport factors, (specifically karyopherin beta), and nuclear transport substrates. Its cytoplasmic aspect implies a role for NUP159 in nuclear import.
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The Essential Yeast Nucleoporin NUP159 Is Located on the Cytoplasmic Side of the Nuclear Pore Complex and Serves in Karyopherin-mediated Binding of Transport Substra
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... An essential yeast nucleoporin, NUP159, has been localized to the cytoplasmic side of the NPC. [38] It contains a coiled-coil domain as well as a domain of repeated motifs. [38] Kraemer et al. [38] used Escherichia coli to express five separate segments of NUP159. ...
... [38] It contains a coiled-coil domain as well as a domain of repeated motifs. [38] Kraemer et al. [38] used Escherichia coli to express five separate segments of NUP159. They found that the segment containing the repeat motif was directly involved in binding a transportable nuclear substrate in the presence of vertebrate cytosolic extract containing NTFs. ...
... [38] It contains a coiled-coil domain as well as a domain of repeated motifs. [38] Kraemer et al. [38] used Escherichia coli to express five separate segments of NUP159. They found that the segment containing the repeat motif was directly involved in binding a transportable nuclear substrate in the presence of vertebrate cytosolic extract containing NTFs. ...
[Review] The nuclear pore complex: A comprehensive review of structure and function
Article
Full-text available
  • Apr 2016
The nuclear pore complex (NPC) is an important functional entity of every eukaryotic cell’s nuclear membrane. It enables selective transport of materials across the nuclear membrane in an organized and orderly fashion. Substances carried in and out of the nucleus by the NPC include three major groups of molecules: (a) Messenger ribonucleic acid molecules, (b) proteins, and (c) ribonucleoproteins (RNPs). The transport across the nuclear membrane involves adenosine triphosphate hydrolysis in the great majority of cases even though certain guanosine triphosphate‑hydrolyzing mechanisms have also been identified. The understanding of the NPC appears crucial to our understanding of certain pathological processes. For example, it has been found that certain human viruses can “trick” our cells into transporting their RNPs into the nucleus using signal peptides similar to the human nuclear‑localizing signals. The major challenge of today’s research on the NPC resides in identification of over one hundred of its distinct polypeptide units and in determining their functions and interactions. To date, many of the structural proteins involved in the NPC have been identified, but the mechanism of their interactions still remains largely hypothetical. This project discusses the structure and function of the NPC. The following core competencies are addressed in this article: Medical knowledge.
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... Interestingly, the yeast dynein light chain Dyn2, which is recruited by Nup159 to the nuclear pores, is another constituent of this subcomplex [18]. Nup159 structure is characterized by several well-defined domains, including an N-terminal β-propeller region that it is essential for nucleocytoplasmic mRNA transport, a central array of FG-rich repeat sequences, a dynein interaction domain (DID) that concentrates 5 consecutive Dyn2-binding motifs, and an α-helical C-region that plays an important role in nuclear pore anchoring, mRNA transport, and Nup159 protein stability [18,[43][44][45]. To analyze whether the C-terminal domain of Nup159 could mediate its association with Bfa1, we used the nup159-1 allele, which encodes a highly unstable protein lacking the last 96 aa of Nup159 [46]. ...
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Article
Full-text available
Both the spindle microtubule-organizing centers and the nuclear pore complexes (NPCs) are convoluted structures where many signaling pathways converge to coordinate key events during cell division. Interestingly, despite their distinct molecular conformation and overall functions, these structures share common components and collaborate in the regulation of essential processes. We have established a new link between microtubule-organizing centers and nuclear pores in budding yeast by unveiling an interaction between the Bfa1/Bub2 complex, a mitotic exit inhibitor that localizes on the spindle pole bodies, and the Nup159 nucleoporin. Bfa1/Bub2 association with Nup159 is reduced in metaphase to not interfere with proper spindle positioning. However, their interaction is stimulated in anaphase and assists the Nup159-dependent autophagy pathway. The asymmetric localization of Bfa1/Bub2 during mitosis raises the possibility that its interaction with Nup159 could differentially promote Nup159-mediated autophagic processes, which might be relevant for the maintenance of the replicative lifespan.
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... The Nup82 complex, also located at the cytoplasmic entrance of the NPC, is another cytoplasmic NPC structure known to be predominantly involved in mRNA export [139,[142][143][144][145] in a way that depends on the relative position of FG repeats in the complex [63] as the Mex67/Mtr2 heterodimer directly engages with these repeats [61,146]. Recent work highlights the role of the Nup84 subcomplex as the Dbp5 anchoring platform [147] (Figure 4B). ...
The Great Escape: mRNA Export through the Nuclear Pore Complex
Article
Full-text available
  • Oct 2021
  • INT J MOL SCI
Nuclear export of messenger RNA (mRNA) through the nuclear pore complex (NPC) is an indispensable step to ensure protein translation in the cytoplasm of eukaryotic cells. mRNA is not translocated on its own, but it forms ribonuclear particles (mRNPs) in association with proteins that are crucial for its metabolism, some of which; like Mex67/MTR2-NXF1/NXT1; are key players for its translocation to the cytoplasm. In this review, I will summarize our current body of knowledge on the basic characteristics of mRNA export through the NPC. To be granted passage, the mRNP cargo needs to bind transport receptors, which facilitate the nuclear export. During NPC transport, mRNPs undergo compositional and conformational changes. The interactions between mRNP and the central channel of NPC are described; together with the multiple quality control steps that mRNPs undergo at the different rings of the NPC to ensure only proper export of mature transcripts to the cytoplasm. I conclude by mentioning new opportunities that arise from bottom up approaches for a mechanistic understanding of nuclear export.
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... Here, we studied the localization of Nup159, Alg1, Tcb3 and Spf1 relative to LDs in yeast exposed to ER stressors. Nup159 is a nuclear pore complex component that is found on the cytosolic face of the nuclear pore and therefore associated with nER [37]. Alg1 is an integral ER membrane protein and mannosyltransferase that is required for asparaginelinked glycosylation [38]. ...
Membrane dynamics and protein targets of lipid droplet microautophagy during ER stress-induced proteostasis in the budding yeast, Saccharomyces cerevisiae
Article
Full-text available
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... Close homologs of Nup146 in budding yeast (Nup159; referred to here as Sc Nup159) and 194 humans (Nup214; referred to as Hs Nup214) are both located exclusively at the cytoplasmic 195 face of NPCs (Gorsch et al., 1995, Kraemer et al., 1994, Kraemer et al., 1995, and indirect ...
Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore
Article
Full-text available
  • May 2018
  • eLife
ELife digest Nearly all cells contain networks of filaments called microtubules that play many different roles. They provide internal structure; they serve as ‘tracks’ for transporting materials from one region of the cell to another; and they help to separate chromosomes during cell division. To understand how microtubules work, it is important to know how they are organized and distributed in cells. In several types of cells, including muscle cells in humans, and most plant cells, microtubules form on the surface of the cell nucleus – the membrane-bound compartment that stores genetic information. A key protein involved in microtubule formation, called Mto1, is present on the surface of the nucleus, but it was not clear how Mto1 localizes there. Using fission yeast cells, Bao et al. have devised a new method to identify the proteins that recruit Mto1 to the surface of the nucleus. This revealed that Mto1 is recruited to the nuclear pores – large channels on the surface of the nucleus through which proteins can be transported. Unexpectedly, the proteins that recruit Mto1 to the nuclear pores do so using a mechanism that is also used to transport proteins out of the nucleus. Thus, in addition to determining how microtubules are organized at the cell nucleus, Bao et al. have identified a previously unknown role for the ‘nuclear transport machinery’. Currently, drugs that inhibit the nuclear transport machinery form potential treatments for some cancers and viral infections. A better understanding of the multiple roles performed by the nuclear transport machinery may help researchers to design more effective inhibitor drugs.
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... . Overproduced and endogenous Nup82-Nup159FG-Nsp1C-Dyn2 complexes have a similar gel filtration behavior and a related MALS value. (A) Expression levels of the indicated Nup159 wild-type (WT) and mutant proteins used in this study, which were analyzed by SDS-PAGE of whole-cell lysates (WCL; cells were grown at 30°C) followed by Western blotting using a monoclonal anti-Nup159 antibody (mAB165C10), which allows detection of the Nup159 CTD constructs (Kraemer et al., 1995), and as loading control, we used anti-Arc1 antibodies. (B) SEC-MALS analysis of the overproduced Nup82-Flag-Nup159FG-Nsp1C-Dyn2 complex with a deduced molecular mass of 632 kD. ...
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The Cell Nucleus and Its Compartments
Chapter
  • Oct 2020
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Ran GTPASE Regulation of the CRM1-Dependent Export Pathway
Chapter
  • Jan 2001
Nuclear proteins that are not stably bound to intranuclear structures have the potential to be transported to the cytoplasm. Recent studies on proteins that undergo nuclear export led to the identification of cis-acting nuclear export signals or NES. These short sequence motifs are recognized by specific receptors, called karyopherins or exportins, which belong to a large family of proteins conserved through evolution. The NES, which in both cellular and viral proteins is enriched in hydrophobic amino acids including leucine, specifically interacts with the nuclear export receptor CRM1. The function of CRM1 is to mediate association with the nuclear pore complex and translocation of the NES protein to the cytoplasm. Binding of NES to CRM1 occurs in a Ran-GTP-dependent manner, but GTP hydrolysis is not required for this binding, or for translocation of the export complex through the NPC. CRM1 and Ran are sufficient to promote translocation of the cargo from the nucleoplasm to the cytoplasmic face of the nuclear pore complex, however, additional Ran·TP-binding proteins including the newly described NXT1 protein are also required to facilitate nuclear export. In the cytoplasm, dissociation of Ran from the export complex is triggered by the concerted action of different Ran regulatory proteins. These include the Ran GTPase Activating Protein, Ran Binding Proteins 1 and 2, and NXT1. Ran dissociation reverses the interactions between NES and CRM1, and CRM1 and the nuclear pore complex. Thus, disassembly of the nuclear export complex leads to the release of the NES-containing cargo in the cytoplasm and the recycling of CRM 1 back to the nucleus where it is available for a new round of transport. In this review, we describe what is known about the various steps in nuclear export of proteins and RNA-protein complexes by the CRM 1-dependent pathway, with special emphasis on the role of the Ran GTPase and associated proteins in this process.
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Cellular Structures and Nucleocytoplasmic Transport
Chapter
  • Jan 1996
‘Well-characterized’ has no precise or stable agreed denotation; its semantic content, if any, derives from context. Generally it carries the connotation that good science has been done and progress has been made. Conversely, ‘X is poorly-characterized’ implies that our knowledge does not provide an adequate basis for claims that might be advanced about X; it usually heralds a skeptical assessment of someone else’s work. Sometimes these phrases are used emotively, serving only laudatory or pejorative functions. So much for precision; the point about stability is even more obvious. In the 1950s an enzyme was well-characterized if it had been purified biochemically and its kinetic properties and inhibitor sensitivities had been quantified; nowadays, sequence data, three-dimensional structure, active site chemistry and regulation of expression seem to be minimal requirements and kinetic information is of secondary interest. A method is well-characterized if it yields results that are judged reproducible, valid and interpretable; but as new methods supersede old, presumably a once-accepted procedure becomes at least relatively ill-characterized. Fashions in scientific method come and go, and as we have already seen this makes it dangerously tempting to dismiss work based on ‘outdated’ methods. Perhaps it is worth recalling that modern astronomers do not disregard Kepler, Herschel or Hubble merely because techniques have improved since then.
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Full-text available
  • Dec 1990
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A new family of yeast nuclear pore complex proteins
Article
  • Nov 1992
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POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope
Article
  • Apr 1994
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Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex
Article
  • Dec 1994
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Gorlich D, Prehn S, Laskey RA, Hartmann EIsolation of a protein that is essential for the first step of nuclear protein import. Cell 79: 767-778
Article
  • Dec 1994
  • CELL
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Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor [published erratum appears in J Cell Biol 1994 Jan;124(1-2):217]
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  • Dec 1993
We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin-permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysis. GTP gamma S and other nonhydrolyzable analogues of GTP were found to rapidly inhibit the rate of in vitro nuclear import. Transport inhibition by GTP gamma S was dependent on the concentrations of permeabilized cells and cytosol, and was strongly enhanced by a cytosolic factor(s). The predominant cytosolic component responsible for this inhibition was found in a 20-30-kD fraction in molecular sieving chromatography. Furthermore, a component(s) of this 20-30-kD fraction was itself required for efficient nuclear import. Biochemical complementation with bacterially expressed protein demonstrated that this essential GTP gamma S-sensitive transport factor was Ran/TC4, a previously described GTPase of the Ras superfamily found in both nucleus and cytoplasm. Ran/TC4 and its guanine nucleotide release protein RCC1 have previously been implicated in DNA replication, cell cycle checkpoint control, and RNA synthesis, processing and export. Our results suggest that Ran/TC4 serves to integrate nuclear protein import with these other nuclear activities.
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The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors
Article
  • Jul 1992
  • CELL
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Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers
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A set of plasmids was constructed that contain the yeast selectable markers HIS3, LEU2, TRP1 or URA3 embedded in the multiple cloning site of pUC18.
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