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E C Hurt

Publications and source records attributed to E C Hurt.

At least 19 recordsLinked to original sources
PubMed1999

Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA.

Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.

Base Sequence↗
PubMed1998

Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes.

The nuclear pore complex (NPC), a supramolecular assembly of approximately 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the approximately 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.

Animals↗
PubMed1998

Cse1p functions as the nuclear export receptor for importin alpha in yeast.

CSE1 is essential for yeast cell viability and has been implicated in chromosome segregation. Based on its sequence similarity, Cse1p has been grouped into the family of importin beta-like nucleocytoplasmic transport receptors with highest homology to the recently identified human nuclear export receptor for importin alpha, CAS. We demonstrate here that Cse1p physically interacts with yeast Ran and yeast importin alpha (Srp1p) in the yeast two-hybrid system and that recombinant Cse1p, Srp1p and Ran-GTP form a trimeric complex in vitro. Re-export of Srp1p from the nucleus into the cytoplasm and nuclear uptake of a reporter protein containing a classical NLS are inhibited in a cse1 mutant strain. These findings suggest that Cse1p is the exportin of importin alpha in yeast.

Cell Nucleus↗
PubMed1998

Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of psi 38 and psi 39 in tRNA anticodon loop.

The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of pseudouridines psi 38 and psi 39 in tRNA whereas the other activities, specific for psi formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected. Also, the His6-tagged recombinant yeast Deg1p expressed in E. coli as well as a protein fusion with protein A in yeast display the enzymatic activity only toward psi 38 and psi 39 formation in different tRNA substrates. Therefore, Deg1p is the third tRNA:pseudouridine synthase (Pus3p) characterized so far in yeast. Disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 degrees C). Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of psi 38- and psi 39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:pseudouridine synthase truA from E. coli (initially called hisT or PSU-I gene product).

Amino Acid Sequence↗
PubMed1998

A conserved domain within Arc1p delivers tRNA to aminoacyl-tRNA synthetases.

Two yeast enzymes that catalyze aminoacylation of tRNAs, MetRS and GluRS, form a complex with the protein Arc1p. We show here that association of Arc1p with MetRS and GluRS is required in vivo for effective recruitment of the corresponding cognate tRNAs within this complex. Arc1p is linked to MetRS and GluRS through its amino-terminal domain, while its middle and carboxy-terminal parts comprise a novel tRNA-binding domain. This results in high affinity binding of cognate tRNAs and increased aminoacylation efficiency. These findings suggest that Arc1p operates as a mobile, trans-acting tRNA-binding synthetase domain and provide new insight into the role of eukaryotic multimeric synthetase complexes.

Amino Acyl-tRNA Synthetases↗
PubMed1997

In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p.

The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.

Binding Sites↗
PubMed1996

NBP35 encodes an essential and evolutionary conserved protein in Saccharomyces cerevisiae with homology to a superfamily of bacterial ATPases.

We have cloned a novel and essential gene, NBP35, from Saccharomyces cerevisiae that encodes a putative Nucleotide Binding Protein of 35 kDa. Sequence analysis revealed structural homology of Nbp35p with a family of bacterial ATPases involved in cell division processes and chromosome partitioning. A search in databases identified closely related sequences from yeast and higher eukaryotes, suggesting a conserved function for this family of proteins. By indirect immunofluorescence, a tagged version of Nbp35p carrying two immunoglobulin G-binding domains derived from Staphylococcus aureus Protein A was localised to the nucleus. A single amino-acid substitution in the conserved nucleotide-binding motif of Nbp35p renders the protein non-functional. Furthermore, a conserved cluster of four cysteines in the N-terminal end of the protein is also required for an essential role of Nbp35p.

Adenosine Triphosphatases↗
PubMed1996

The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases.

Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore-associated yeast protein involved in tRNA biogenesis. Arc1p is associated with two proteins which were identified as methionyl-tRNA and glutamyl-tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method. ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1- mutants are complemented by the genes for MetRS and GluRS. Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N-terminal truncated form, and strongly increases its apparent affinity for tRNAMet. Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV-cross-linking. Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl-tRNA synthetases. This functional interaction may be required for efficient aminoacylation in vivo.

Acylation↗
PubMed1996

Nuclear pore proteins are involved in the biogenesis of functional tRNA.

Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA.

Amino Acid Sequence↗
PubMed1996

A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores.

In a genetic screen for nucleoporin-interacting components, a novel nuclear pore protein Nup84p, which exhibits homology to mammalian Nup107p, was isolated. Nup84p forms a complex with five proteins, of which Nup120p, Nup85p, Sec13p, and a Sec13p homolog were identified. Upon isolation of Sec13p-ProtA, nucleoporins were still associated, but the major copurifying band was a 150 kDa protein, showing that Sec13p occurs in two complexes. Disruption of any of the genes encoding Nup84p, Nup85p, or Nup120p caused defects in nuclear membrane and nuclear pore complex organization, as well as in poly(A)+ RNA transport. Thus, the Nup84p complex in conjunction with Sec13-type proteins is required for correct nuclear pore biogenesis.

Amino Acid Sequence↗
PubMed1996

Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p.

The amino-terminal domain of Nic96p physically interacts with the Nsp1p complex which is involved in nucleocytoplasmic transport. Here we show that thermosensitive mutations mapping in the central domain of Nic96p inhibit nuclear pore formation at the nonpermissive temperature. Furthermore, the carboxyterminal domain of Nic96p functionally interacts with a novel nucleoporin Nup188p in an allele-specific fashion, and when ProtA-Nup188p was affinity purified, a fraction of Nic96p was found in physical interaction. Although NUP188 is not essential for viability, a null mutant exhibits striking abnormalities in nuclear envelope and nuclear pore morphology. We propose that Nic96p is a multivalent protein of the nuclear pore complex linked to several nuclear pore proteins via its different domains.

Amino Acid Sequence↗
PubMed1996

Yeast nucleoporin mutants are defective in pre-tRNA splicing.

We have screened nucleoporin mutants for the inhibition of tRNA splicing, which has previously been proposed to be coupled to transport. Strains mutant for Nup49p or Nup116p, or genetically depleted of Nup145p, strongly accumulated unspliced pre-tRNAs. Splicing was inhibited for all 10 families of intron-containing pre-tRNA, but no effects on 5' or 3' end processing were detected. Strains mutant for Nup133p or Nsp1p accumulated lower levels of several unspliced pre-tRNAs. In contrast, no accumulation of any pre-tRNA was observed in strains mutant for Nup1p, Nup85p, or Nup100p. Other RNA processing reactions tested, pre-rRNA processing, pre-mRNA splicing, and small nucleolar and small nuclear RNA synthesis, were not clearly affected for any nucleoporin mutant. These data provide evidence for a coupling between pre-tRNA splicing and nuclear-cytoplasmic transport. Mutation of NUP49, NUP116, or NUP145 has previously been shown to lead to nuclear poly(A)+ RNA accumulation, indicating that these nucleoporins play roles in the transport of more than one class of RNA.

Base Sequence↗
PubMed1995

Nucleocytoplasmic transport: factors and mechanisms.

In the past two years, our knowledge concerning the mechanisms of nucleocytoplasmic transport through the nuclear pore complex (NPC) has considerably expanded. The application of in vitro systems that reconstitute nuclear protein import has allowed the identification of cytosolic factors that are required for the import process. Microinjection into Xenopus oocytes and yeast genetic systems have provided interesting candidates for RNA export mediators. Functional and structural analysis of nucleoporins has demonstrated the involvement of NPC components in the transport process. Finally, new concepts have emerged such as the integration of the mechanisms of the nuclear protein import and RNA export reactions and the assembly of the transport machinery at specialised domains of the NPC.

Amino Acid Sequence↗
PubMed1995

Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p.

Nic96p has been isolated previously in a complex together with the nuclear pore proteins Nsp1p, Nup49p and a p54 polypeptide. In a genetic screen for Nsp1p-interacting components, we now find NIC96, as well as a novel gene NUP57 which encodes the p54 protein (called Nup57p). Nup57p which is essential for cell growth contains GLFG repeats in the N-terminal half and heptad repeats in the C-terminal half. The domain organization of Nic96p is more complex: N-terminally located heptad repeats mediate binding to a trimeric Nsp1p-Nup49p-Nup57p complex, but are not required for the formation of this core complex; single amino acid substitutions in the central domain yield thermosensitive mutants, which do not impair interaction with the Nsp1 complex; the C-terminal domain is neither essential nor required for binding to the nucleoporin complex, but strikingly mutations in this part cause synthetic lethality with nsp1 and nup57 mutant alleles. Since a strain in which the Nic96p heptad repeats were deleted shows, similar to nsp1 and nup49 mutants, cytoplasmic mislocalization of a nuclear reporter protein, we propose that the interaction of the heterotrimeric Nsp1p-Nup49p-Nup57p core complex with Nic96p is required for protein transport into the nucleus.

Amino Acid Sequence↗
PubMed1995

Genetic approaches to nuclear pore structure and function.

The major features of nucleocytoplasmic transport through nuclear pore complexes are conserved from yeast to humans. This transport machinery, which includes the 125 MDa nuclear pore complex structure, has been molecularly dissected in the yeast Saccharomyces cerevisiae by genetic approaches. Here, we summarize the genetic analyses used to elucidate structure-function relationships within this large supramolecular assembly.

Antibodies, Monoclonal↗
PubMed1995

A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p.

Nsp1p interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p in a stable complex which participates in nucleocytoplasmic transport. An additional p80 component is associated with Nsp1p, but does not co-purify with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and encodes a novel essential nuclear pore protein named Nup82p. Immunoprecipitation of tagged Nup82p reveals that it is physically associated with a fraction of Nsp1p which is distinct from Nsp1p found in a complex with Nup57p, Nic96p and Nup49p. The Nup82 protein can be divided into at least two different domains both required for the essential function, but it is only the carboxy-terminal domain, exhibiting heptad repeats, which binds to Nsp1p. Yeast cells depleted of Nup82p stop cell growth and concomitantly show a defect in poly(A)+RNA export, but no major alterations of nuclear envelope structure and nuclear pore density are seen by EM. This shows that Nsp1p participates in multiple interactions at the NPC and thus has the capability to physically interact with different NPC structures.

Amino Acid Sequence↗