Biomedical subjects

E Hurt

Publications and source records attributed to E Hurt.

At least 37 records · Page 2Linked to original sources

PubMed2000

Yra1p, a conserved nuclear RNA-binding protein, interacts directly with Mex67p and is required for mRNA export.

Mex67p and Mtr2p constitute an essential mRNA export complex that interacts with poly(A)+ RNA and nuclear pore proteins. We have identified Yra1p, an intranuclear protein with in vitro RNA-RNA annealing activity, which directly binds to Mex67p. The complex between Yra1p and Mex67p was reconstituted in vitro and shown by UV-crosslinking to bind directly to RNA. Mutants of YRA1 are impaired in nuclear poly(A)+ RNA export at restrictive growth conditions. ALY, the mouse homologue of Yra1p and a transcriptional coactivator, can bind in vitro to yeast and human Mex67p and partly complements the otherwise non-viable yra1 null mutant. Thus, Yra1p is the first RNA-binding protein characterized, which bridges the shuttling Mex67p/Mtr2p exporter to intranuclear mRNA transport cargoes.

Amino Acid Sequence↗

PubMed2000

Mlp2p, a component of nuclear pore attached intranuclear filaments, associates with nic96p.

A fraction of the yeast nucleoporin Nic96p is localized at the terminal ring of the nuclear basket. When Nic96p was affinity purified from glutaraldehyde-treated spheroplasts, it was found to be associated with Mlp2p. Mlp2p, together with Mlp1p, are the yeast Tpr homologues, which form the nuclear pore-attached intranuclear filaments (Strambio-de-Castillia, C., Blobel, G., and Rout, M. P. (1999) J. Cell Biol. 144, 839-855). Double disruption mutants of MLP1 and MLP2 are viable and apparently not impaired in nucleocytoplasmic transport. However, overproduction of MLP1 causes nuclear accumulation of poly(A)(+) RNA in a chromatin-free area of the nucleus.

Amino Acid Sequence↗

PubMed2000

Purification of protein A-tagged yeast ran reveals association with a novel karyopherin beta family member, Pdr6p.

The small GTPase Ran (encoded by GSP1 and GSP2 in yeast) plays a central role in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant. After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin beta transport receptors (e.g. Kap121p and Kap123p) and a novel member of this protein family, Pdr6p, were found to be associated with yeast Ran. Subsequent tagging of Pdr6p with green fluorescent protein revealed association with the nuclear pore complexes in vivo. Thus, functional tagging of yeast Ran allowed the study of its in vivo distribution and interaction with known and novel Ran-binding proteins.

Cell Compartmentation↗

PubMed2000

Review: transport of tRNA out of the nucleus-direct channeling to the ribosome?

Although tRNA was the first substrate whose export from the nuclei of eukaryotic cells had been shown to be carrier-mediated and active, it has only been in the last 2 years that the first mechanistic details of this nucleocytoplasmic transport pathway have begun to emerge. A member of the importin/karyopherin beta superfamily, Los1p in yeast and Xpo-t in vertebrates, has been shown to export tRNA in cooperation with the small GTPase Ran (Gsp1p) from the nucleus into the cytoplasm, where tRNA becomes available for translation. However, Los1p is not essential for viability in yeast cells, suggesting that alternative tRNA export pathways exist. Recent results show that aminoacylation and a translation factor are also required for efficient nuclear tRNA export. Thus, protein translation and nuclear export of tRNA appear to be coupled processes.

Animals↗

PubMed2000

Yeast nuclear pore complex assembly defects determined by nuclear envelope reconstruction.

Assembly of nuclear pore complexes (NPCs) is a critical yet poorly understood cellular function. One approach to studying NPC assembly is to identify yeast mutants defective in this process. This requires robust assays for NPC assembly that can be used for phenotypic analysis. We have previously reconstructed yeast nuclei from electron micrographs of serially sectioned cells to precisely determine the number of NPCs (Winey et al., 1997). Here we report the analysis of strains mutant in either of two nucleoporin-encoding genes, NIC96 (Zabel et al., 1996) and NUP192 (Kosova et al., 1999). Using conditional alleles of either gene, we have found that the NPC number falls significantly following shift to the restrictive temperature. We conclude that the drop in NPC number results from the failure to assemble new NPCs during cell divisions, leading to the dilution of NPCs that existed when the cells were shifted to the restrictive temperature. We are also able to document a subtle defect in NPC numbers in nup192-15 cells at their permissive temperature. The data presented here quantitatively demonstrate that NPC numbers fall in nic96-1 and nup192-15 strains upon shifting to the restrictive temperature, indicating that these gene products are required for NPC assembly.

Fungal Proteins↗

PubMed2000

Yeast Ran-binding protein 1 (Yrb1) shuttles between the nucleus and cytoplasm and is exported from the nucleus via a CRM1 (XPO1)-dependent pathway.

The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo.

Animals↗

PubMed1999

Nup192p is a conserved nucleoporin with a preferential location at the inner site of the nuclear membrane.

Human Nup93, the homologue of yeast Nic96p, is associated with a 205-kDa protein whose intracellular location and function is unknown. We show here that the yeast open reading frame YJL039c, which is homologous to this human p205, encodes the so far largest yeast nucleoporin. Accordingly, green fluorescent protein (GFP)-tagged YJL039c was localized to the nuclear pores and therefore named Nup192p. Affinity purification of ProtA-Nic96p from glutaraldehyde-fixed spheroplasts reveals association with Nup192p. NUP192 is essential for cell growth. A temperature-sensitive mutant nup192-15 is neither impaired in nuclear import of a SV40 nuclear localization sequence-containing reporter protein nor in mRNA export, but association of Nup49-GFP with nuclear pores is inhibited at the non-permissive temperature. By immunoelectron microscopy, Nup192p-ProtA is seen at the inner site of the nuclear pores, at a distance of 60 +/- 15 nm from the central plane of the pore. This suggests that Nup192p is an evolutionarily conserved structural component of the nuclear pore complex with a preferential location at the inner site of the nuclear membrane.

Amino Acid Sequence↗

PubMed1999

Nuclear RNA export in yeast.

Eukaryotic cells massively exchange macromolecules (proteins and RNAs) between the nucleus and cytoplasm through the nuclear pore complexes. Whereas a mechanistic picture emerges of how proteins are imported into and exported from the nucleus, less is known about nuclear exit of the different classes of RNAs. However, the yeast Saccharomyces cerevisiae offers an experimental system to study nuclear RNA export in vivo and thus to genetically dissect the different RNA export machineries. In this review, we summarize our current knowledge and recent progress in identifying components involved in nuclear RNA export in yeast.

Animals↗

PubMed1999

The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human.

Human TAP is an orthologue of the yeast mRNA export factor Mex67p. In mammalian cells, TAP has a preferential intranuclear localization, but can also be detected at the nuclear pores and shuttles between the nucleus and the cytoplasm. TAP directly associates with mRNA in vivo, as it can be UV-crosslinked to poly(A)+ RNA in HeLa cells. Both the FG-repeat domain of nucleoporin CAN/Nup214 and a novel human 15 kDa protein (p15) with homology to NTF2 (a nuclear transport factor which associates with RanGDP), directly bind to TAP. When green fluorescent protein (GFP)-tagged TAP and p15 are expressed in yeast, they localize to the nuclear pores. Strikingly, co-expression of human TAP and p15 restores growth of the otherwise lethal mex67::HIS3/mtr2::HIS3 double knockout strain. Thus, the human TAP-p15 complex can functionally replace the Mex67p-Mtr2p complex in yeast and thus performs a conserved role in nuclear mRNA export.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Transfer RNA biogenesis: A visa to leave the nucleus.

Only correctly folded and mature tRNAs can leave the nucleus and enter the cytoplasm. Surprisingly, tRNA-aminoacylation has been found to occur, not only in the cytosol, but also inside the nucleus, where it may act as an additional proofreading step and facilitate the export of 'ready-to-function' aminoacyl-tRNAs.

Animals↗

PubMed1999

A novel in vivo assay reveals inhibition of ribosomal nuclear export in ran-cycle and nucleoporin mutants.

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.

Biological Transport, Active↗

PubMed1998

A novel complex of membrane proteins required for formation of a spherical nucleus.

Two membrane proteins were identified through their genetic interaction with the nucleoporin Nup84p and shown to participate in nuclear envelope morphogenesis in yeast. One component is a known sporulation factor Spo7p, and the other, Nem1p, a novel protein whose C-terminal domain is conserved during eukaryotic evolution. Spo7p and Nem1p localize to the nuclear/ER membrane and behave biochemically as integral membrane proteins. Nem1p binds to Spo7p via its conserved C-terminal domain. Although cells without Spo7p or Nem1p are viable, they exhibit a drastically altered nuclear morphology with long, pore-containing double nuclear membrane extensions. These protrusions emanate from a core nucleus which contains the DNA, and penetrate deeply into the cytoplasm. Interestingly, not only Spo7(-) and Nem1(-), but also several nucleoporin mutants are defective in sporulation. Thus, Spo7p and Nem1p, which exhibit a strong genetic link to nucleoporins of the Nup84p complex, fulfil an essential role in formation of a spherical nucleus and meiotic division.

Amino Acid Sequence↗

PubMed1998

Mtr10p functions as a nuclear import receptor for the mRNA-binding protein Npl3p.

MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)-tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)-containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non-repetitive C-terminal tail. Mtr10p interacts in vitro with Gsp1p-GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran-GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran-GTP and newly synthesized mRNA.

Amino Acid Sequence↗

PubMed1998

Nup116p and nup100p are interchangeable through a conserved motif which constitutes a docking site for the mRNA transport factor gle2p.

Nup116p and Nup100p are highly related yeast GLFG nucleoporins, but only Nup116p is stoichiometrically bound to Gle2p, a previously identified mRNA export factor. A short Gle2p-binding sequence within Nup116p (GLEBS; residues 110-166) is sufficient and necessary to anchor Gle2p at the nuclear pores, whereas the carboxy-terminal domain of Nup116p mediates its own nuclear pore complex (NPC) association. The GLEBS is evolutionarily conserved and found in rat/Xenopus Nup98 and an uncharacterized Caenorhabditis elegans ORF, but is absent from Nup100p. When the GLEBS is deleted from Nup116p, Gle2p dissociates from the nuclear envelope and clusters of herniated nuclear pores form. When the GLEBS is inserted into Nup100p, Nup100p-GLEBS complements both the thermosensitive and NPC-herniated phenotype of nup116- cells, and Gle2p is retargeted concomitantly to the NPCs. Thus, the in vivo function of Gle2p is strictly coupled to the short GLEBS within Nup116p which links this putative mRNA transport factor to the nuclear pores.

Amino Acid Sequence↗

PubMed1998

The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity.

We have previously shown that the yeast gene PUS1 codes for a tRNA:pseudouridine synthase and that recombinant Pus1p catalyzes, in an intron-dependent way, the formation of psi34 and psi36 in the anticodon loop of the yeast minor tRNA(Ile) in vitro (Simos G et al., 1996, EMBO J 15:2270-2284). Using a set of T7 transcripts of different tRNA genes, we now demonstrate that yeast pseudouridine synthase 1 catalyzes in vitro pseudouridine formation at positions 27 and/or 28 in several yeast cytoplasmic tRNAs and at position 35 in the intron-containing tRNA(Tyr) (anticodon GUA). Thus, Pus1p not only displays a broad specificity toward the RNA substrates, but is also capable of catalyzing the pseudouridine (psi) formation at distinct noncontiguous sites within the same tRNA molecule. The cell-free extract prepared from the yeast strain bearing disrupted gene PUS1 is unable to catalyze the formation of psi27, psi28, psi34, and psi36 in vitro, however, psi35 formation in the intron-containing tRNA(Tyr)(GUA) remains unaffected. Thus, in yeast, only one gene product accounts for tRNA pseudouridylation at positions 27, 28, 34, and 36, whereas for position 35 in tRNA(Tyr), another site-specific tRNA:pseudouridine synthase with overlapping specificity exists. Mapping of pseudouridine residues present in various tRNAs extracted from the PUS1-disrupted strain confirms the in vitro data obtained with the recombinant Pus1p. In addition, they suggest that Pus1p is implicated in modification at positions U26, U65, and U67 in vivo.

Cloning, Molecular↗

PubMed1998

Yeast Los1p has properties of an exportin-like nucleocytoplasmic transport factor for tRNA.

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-beta-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.

Fungal Proteins↗

PubMed1998

Nuclear mRNA export requires complex formation between Mex67p and Mtr2p at the nuclear pores.

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.

Escherichia coli↗

PubMed1997

Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins.

Nup145p is an essential yeast nucleoporin involved in nuclear export of polyadenylated RNAs. We demonstrate here that Nup145p is cleaved in vivo to yield two functionally distinct domains: a carboxy-terminal domain (C-Nup145p) which is located at the nuclear pore complex (NPC) and assembles into the Nup84p complex, and a GLFG-containing amino-terminal domain (N-Nup145p) which is not part of this complex. Whereas the essential C-Nup145p accomplishes the functions required for efficient mRNA export and normal NPC distribution, N-Nup145p, which is homologous to the GLFG-containing nucleoporins Nup100p and Nup116p, is not necessary for cell growth. However, the N-Nup145p becomes essential in a nup188 mutant background. Strikingly, generation of a free N-domain is a prerequisite for complementation of this peculiar synthetic lethal mutant. These data suggest that N- and C-domains of Nup145p perform independent functions, and that the in vivo cleavage observed is of functional importance.

Amino Acid Sequence↗