Mutations in theYRB1Gene Encoding Yeast Ran-Binding-Protein-1 That Impair Nucleocytoplasmic Transport and Suppress Yeast Mating Defects

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1089-1105 ◽  
Author(s):  
Markus Künzler ◽  
Joshua Trueheart ◽  
Claudio Sette ◽  
Eduard Hurt ◽  
Jeremy Thorner
Keyword(s):  
Nuclear Export ◽  
Protein Import ◽  
Essential Gene ◽  
Binding Protein ◽  
Nucleocytoplasmic Transport ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Nucleocytoplasmic Trafficking ◽  
Primary Defect

AbstractWe identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (α-importin) and several β-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Δ and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (9) ◽  
pp. 6350-6360
Author(s):  
F Houman ◽  
C Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Essential Gene ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nonsense Mutations ◽  
Carboxy Terminal

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.

scholarly journals RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

1992 ◽  
Vol 12 (10) ◽  
pp. 4314-4326 ◽  
Author(s):  
C Mann ◽  
J Y Micouin ◽  
N Chiannilkulchai ◽  
I Treich ◽  
J M Buhler ◽  
...  
Keyword(s):  
Cell Division ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Essential Gene ◽  
Temperature Sensitive ◽  
G1 Arrest ◽  
Restrictive Temperature ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Carboxy Terminal

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

Abstract P217: Nuclear Protein Import as the Missing Link in Phenylephrine-Induced Cardiomyocyte Hypertrophy

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Mirna N Chahine ◽  
Maxime Mioulane ◽  
Gabor Földes ◽  
Alexander Lyon ◽  
Sian E Harding
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Translocation ◽  
Cardiomyocyte Hypertrophy ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
Adult Rat ◽  
Nuclear Protein Import

During cardiac hypertrophy, cardiomyocytes (CM) present alterations in gene expression and increased contractile protein content. Nuclear protein import (NPI) is critical in regulating gene expression, transcription, and subsequently cell hypertrophy. However, it is unknown how the nuclear transport machinery (transport receptors and nuclear pore complex (NPC)) functions to sustain increased demands for nucleocytoplasmic trafficking. The aim of this study was to determine if exposure of adult CM to phenylephrine (PE) affects hypertrophy by altering NPI and NPC density. Comparisons were made to adult failing rat and human CM. Rat myocytes were enzymatically isolated from adult hearts, and used for immunocytochemistry, qPCR and western immunoblotting. Failing CM were obtained from explanted human hearts at the time of transplant and from a rat model of myocardial infarction-induced hypertrophy and failure. Rat adult CM exposed for 48h to PE were injected with a protein import substrate (Alexa488-BSA-NLS) to visually monitor nuclear import with the confocal microscope. The effects of P38 MAPK inhibitor, HDAC inhibitor, Exportin-1 (CRM-1) inhibitor, and GSK-3 β inhibitor were investigated. Cell and nuclear sizes were increased in PE treated-adult rat CM and in the adult failing rat and human CM compared to normal CM. In contrast, PE depressed the rate and maximal NPI (by 65 +/- 3.4 % (3.55 from 5.46), p<0.05) as well as nucleoporin p62 mRNA and protein expression levels in adult rat CM compared to non-treated CM. Nucleoporin p62, cytoplasmic Ranbp1, and nuclear translocation of importins (Imp.α and β) relative densities were also decreased in PE treated-adult rat CM and in adult failing rat CM and human heart tissue compared to normal controls. On the contrary, CRM-1 nuclear export relative density was increased during the same pathological conditions. Thus NPI downregulation is linked to an increased nuclear export required by CM to generate the hypertrophic phenotype. All these effects were P38MAPK, HDAC and CRM-1 dependent but GSK-3Beta independent in rat CM. Our results show that alterations in NPI and NPC density occur in failing CM as well as in CM under hypertrophic stimuli. NPI may represent a critical therapeutic target in hypertrophic conditions.

bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans

1991 ◽  
Vol 99 (4) ◽  
pp. 711-719
Author(s):  
K.L. O'Donnell ◽  
A.H. Osmani ◽  
S.A. Osmani ◽  
N.R. Morris
Keyword(s):  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Essential Gene ◽  
Tetratricopeptide Repeat ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reading Frame ◽  
Integrative Transformation

The recessive, temperature-sensitive bimA1 mutation of Aspergillus nidulans blocks nuclei in metaphase at restrictive temperature. To determine whether the bimA product is essential, integrative transformation was used to create a mutation in the bimA gene. The mutation was maintained in a heterokaryon and the phenotype of spores produced by the heterokaryon was analyzed. Molecular disruption of the wild-type bimA gene is recessive in the heterokaryon and causes a metaphase block, demonstrating that bimA is an essential gene for mitosis. bimA was cloned by DNA-mediated complementation of its mutant phenotype at restrictive temperature, and the nucleotide sequence of a full-length cDNA was determined. A single large open reading frame was identified in the cDNA sequence, which predicts a protein containing 806 amino acid residues that is related (30.4% identity) to the Schizosaccharomyces pombe nuc2+ gene product, which also is required for completion of mitosis. The sequence of the bimA gene indicates that it is a member of a family of mostly nuclear proteins that contain a degenerate 34 amino acid repeat, the TPR (tetratricopeptide repeat) gene family.

scholarly journals MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

1993 ◽  
Vol 122 (5) ◽  
pp. 1003-1012 ◽  
Author(s):  
JL Emtage ◽  
RE Jensen
Keyword(s):  
Protein Import ◽  
Early Stage ◽  
Osmotic Shock ◽  
Alkali Treatment ◽  
Mitochondrial Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.

scholarly journals The Nsp1p Carboxy-Terminal Domain Is Organized into Functionally Distinct Coiled-Coil Regions Required for Assembly of Nucleoporin Subcomplexes and Nucleocytoplasmic Transport

2001 ◽  
Vol 21 (23) ◽  
pp. 7944-7955 ◽  
Author(s):  
Susanne M. Bailer ◽  
Carolin Balduf ◽  
Ed Hurt
Keyword(s):  
Protein Import ◽  
Coiled Coil ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nuclear Pores ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

ABSTRACT Nucleoporin Nsp1p, which has four predicted coiled-coil regions (coils 1 to 4) in the essential carboxy-terminal domain, is unique in that it is part of two distinct nuclear pore complex (NPC) subcomplexes, Nsp1p-Nup57p-Nup49p-Nic96p and Nsp1p-Nup82p-Nup159p. As shown by in vitro reconstitution, coiled-coil region 2 (residues 673 to 738) is sufficient to form heterotrimeric core complexes and can bind either Nup57p or Nup82p. Accordingly, interaction of Nup82p with Nsp1p coil 2 is competed by excess Nup57p. Strikingly, coil 3 and 4 mutants are still assembled into the core Nsp1p-Nup57p-Nup49p complex but no longer associate with Nic96p. Consistently, the Nsp1p-Nup57p-Nup49p core complex dissociates from the nuclear pores in nsp1coil 3 and 4 mutant cells, and as a consequence, defects in nuclear protein import are observed. Finally, the nsp1-L640Stemperature-sensitive mutation, which maps in coil 1, leads to a strong nuclear mRNA export defect. Thus, distinct coiled-coil regions within Nsp1p-C have separate functions that are related to the assembly of different NPC subcomplexes, nucleocytoplasmic transport, and incorporation into the nuclear pores.

scholarly journals Altered RNA processing and export lead to retention of mRNAs near transcription sites and nuclear pore complexes or within the nucleolus

2016 ◽  
Vol 27 (17) ◽  
pp. 2742-2756 ◽  
Author(s):  
Biplab Paul ◽  
Ben Montpetit
Keyword(s):  
Single Molecule ◽  
Rna Processing ◽  
Nuclear Export ◽  
Essential Gene ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Eukaryotic Gene ◽  
Transcription Sites ◽  
Pore Complexes

Many protein factors are required for mRNA biogenesis and nuclear export, which are central to the eukaryotic gene expression program. It is unclear, however, whether all factors have been identified. Here we report on a screen of >1000 essential gene mutants in Saccharomyces cerevisiae for defects in mRNA processing and export, identifying 26 mutants with defects in this process. Single-molecule FISH data showed that the majority of these mutants accumulated mRNA within specific regions of the nucleus, which included 1) mRNAs within the nucleolus when nucleocytoplasmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, 2) the buildup of mRNAs near transcription sites in 3′-end processing and chromosome segregation mutants, and 3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data show that alterations to various nuclear processes lead to the retention of mRNAs at discrete locations within the nucleus.

scholarly journals A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III.

1990 ◽  
Vol 10 (12) ◽  
pp. 6123-6131 ◽  
Author(s):  
J Archambault ◽  
K T Schappert ◽  
J D Friesen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Essential Gene ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Gene Encoding ◽  
Temperature Sensitive Mutation

RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4. These results support the idea that the RPO26 and RPO21 gene products interact.

RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest

1992 ◽  
Vol 12 (10) ◽  
pp. 4314-4326
Author(s):  
C Mann ◽  
J Y Micouin ◽  
N Chiannilkulchai ◽  
I Treich ◽  
J M Buhler ◽  
...  
Keyword(s):  
Cell Division ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Essential Gene ◽  
Temperature Sensitive ◽  
G1 Arrest ◽  
Restrictive Temperature ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Carboxy Terminal

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells

2001 ◽  
Vol 114 (19) ◽  
pp. 3565-3574 ◽  
Author(s):  
Akemi Kawai ◽  
Shuh-ichi Nishikawa ◽  
Aiko Hirata ◽  
Toshiya Endo
Keyword(s):  
Protein Synthesis ◽  
Elevated Temperature ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Yeast Cells ◽  
Mitochondrial Morphology ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitochondrial Matrix ◽  
Mitochondrial Hsp70

Ssc1p, a member of the Hsp70 family in the mitochondrial matrix of budding yeast, mediates protein import into mitochondria and prevents irreversible aggregation of proteins in the mitochondrial matrix during folding/assembly or at elevated temperature. Here, we show that functional inactivation of the mitochondrial Hsp70 system causes aggregation of mitochondria. When temperature-sensitive mitochondrial Hsp70 mutant cells were incubated at restrictive temperature, a tubular network of mitochondria was collapsed to form aggregates. Inhibition of protein synthesis in the cytosol did not suppress the mitochondrial aggregation and functional impairment of Tim23, a subunit of mitochondrial protein translocator in the inner membrane, did not cause mitochondrial aggregation. Therefore defects of the Hsp70 function in protein import into mitochondria or resulting accumulation of precursor forms of mitochondrial proteins outside the mitochondria are not the causal reason for the aberrant mitochondrial morphology. By contrast, deletion of Mdj1p, a functional partner for mitochondrial Hsp70 in prevention of irreversible protein aggregation in the matrix, but not in protein import into mitochondria, caused aggregation of mitochondria, which was enhanced at elevated temperature (37°C). The aggregation of mitochondria at 37°C was reversed when the temperature was lowered to 23°C unless protein synthesis was blocked. On the basis of these results, we propose that the mitochondrial matrix contains a protein that is responsible for the maintenance of mitochondrial morphology and requires mitochondrial Hsp70 for its function.

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