scholarly journals The Ccr4-Not Complex and yTAF1 (yTafII130p/yTafII145p) Show Physical and Functional Interactions

2002 ◽  
Vol 22 (19) ◽  
pp. 6735-6749 ◽  
Author(s):  
Cécile Deluen ◽  
Nicole James ◽  
Laurent Maillet ◽  
Miguel Molinete ◽  
Grégory Theiler ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Tata Binding Protein ◽  
Molecular Evidence ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Global Regulator ◽  
Functional Interactions ◽  
Terminal Domain ◽  
Synthetically Lethal ◽  
Promoter Dna

ABSTRACT The Saccharomyces cerevisiae Ccr4-Not complex is a global regulator of transcription that is thought to regulate TATA binding protein (TBP) function at certain promoters specifically. In this paper, we show interactions between the essential domain of Not1p, which interacts with Not4p and Not5p, and the N-terminal domain of yTAF1. We isolated a temperature-sensitive nonsense allele of TAF1, taf1-4, which is synthetically lethal at the permissive temperature when combined with not4 and not5 mutants and which produces high levels of a C-terminally truncated yTAF1 derivative. Overexpression of C-terminally truncated yTAF1 is toxic in not4 or not5 mutants, whereas overexpression of full-length yTAF1 suppresses not4. Furthermore, mutations in the autoinhibitory N-terminal TAND domain of yTAF1 suppress not5, and the overexpression of similar mutants does not suppress not4. We find that, like Not5p, yTAF1 acts as a repressor of stress response element-dependent transcription. Finally, we have evidence for stress-regulated occupancy of promoter DNA by Not5p and for Not5p-dependent regulation of yTAF1 association with promoter DNA. Taken together with our finding that Not1p copurifies with glutathione S-transferase-yTaf1 in large complexes, these results provide the first molecular evidence that the Ccr4-Not complex might interact with yTAF1 to regulate its association at promoters, a function that might in turn regulate the autoinhibitory N-terminal domain of yTAF1.

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 Bni1p Regulates Microtubule-Dependent Nuclear Migration through the Actin Cytoskeleton in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (12) ◽  
pp. 8016-8027 ◽  
Author(s):  
Takeshi Fujiwara ◽  
Kazuma Tanaka ◽  
Eiji Inoue ◽  
Mitsuhiro Kikyo ◽  
Yoshimi Takai
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Nuclear Migration ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Cortical Actin ◽  
Synthetic Lethal ◽  
Downstream Target ◽  
Synthetically Lethal ◽  
Mother Cells

ABSTRACT The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes. They were the previously identifiedPAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150Glued, a component of the dynein-activated dynactin complex. Disruption ofBNI1 in either the pac1 or nip100mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.

scholarly journals The Saccharomyces cerevisiae SDA1 gene is required for actin cytoskeleton organization and cell cycle progression

2000 ◽  
Vol 113 (7) ◽  
pp. 1199-1211
Author(s):  
G. Buscemi ◽  
F. Saracino ◽  
D. Masnada ◽  
M.L. Carbone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Actin Cytoskeleton ◽  
Cell Cycle Progression ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Cycle Progression ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization

The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiae novel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G(1) by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1 temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1 or in cdc28-13 cells released from G(1) block, prevents recovery of budding and DNA replication. We also show that the block in G(1) caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G(1) and that Sda1 is involved in the control of the actin cytoskeleton.

scholarly journals Functional interaction of cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein translocation in vivo.

1996 ◽  
Vol 16 (8) ◽  
pp. 4378-4386 ◽  
Author(s):  
J Becker ◽  
W Walter ◽  
W Yan ◽  
E A Craig
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Genetic Interaction ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Alpha Factor ◽  
Synthetically Lethal ◽  
Shock Proteins ◽  
Related Proteins

In order to analyze the in vivo role of the SSA class of cytosolic 70-kDa heat shock proteins (hsps) of Saccharomyces cerevisiae, we isolated a temperature-sensitive mutant of SSA1. The effect of a shift of mutant cells (ssa1ts ssa2 ssa3 ssa4) from the permissive temperature of 23 degrees C to the nonpermissive temperature of 37 degrees C on the processing of several precursor proteins translocated into the endoplasmic reticulum or mitochondria was assessed. Of three mitochondrial proteins tested, the processing of only one, the beta subunit of the F1F0 ATPase, was dramatically affected. Of six proteins destined for the endoplasmic reticulum, the translocation of only prepro-alpha-factor and proteinase A was inhibited. The processing of prepro-alpha-factor was inhibited within 2 min of the shift to 37 degrees C, suggesting a direct effect of the hsp70 defect on translocation. More than 50% of radiolabeled alpha-factor accumulated in the precursor form, with the remainder rapidly reaching the mature form. However, the translocation block was complete, as the precursor form could not be chased through the translocation pathway. Since DnaJ-related proteins are known to interact with hsp70s and strains containing conditional mutations in a dnaJ-related gene, YDJ1, are defective in translocation of prepro-alpha-factor, we looked for a genetic interaction between SSA genes and YDJ1 in vivo. We found that a deletion mutation of YDJ1 was synthetically lethal in a ssa1ts ssa2 ssa3 ssa4 background. In addition, a strain containing a single functional SSA gene, SSA1, and a deletion of YDJ1 accumulated the precursor form of alpha-factor. However, no genetic interaction was observed between a YDJ1 mutation and mutations in the SSB genes, which encode a second class of cytosolic hsp70 chaperones. These results are consistent with SSA proteins and Ydj1p acting together in the translocation process.

scholarly journals Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae.

1997 ◽  
Vol 8 (4) ◽  
pp. 647-662 ◽  
Author(s):  
F P Finger ◽  
P Novick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitive ◽  
Synthetic Lethal ◽  
Growth Defects ◽  
Actin Structure ◽  
Synthetically Lethal ◽  
Bud Site ◽  
Slow Growing ◽  
Late Stages ◽  
Synthetic Growth

Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8. The new sec3 alleles affect early as well as late stages of secretion. Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1). The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive. All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111. The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1. Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin. All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4. This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis.

scholarly journals Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum.

1991 ◽  
Vol 11 (11) ◽  
pp. 5727-5734 ◽  
Author(s):  
C d'Enfert ◽  
C Barlowe ◽  
S Nishikawa ◽  
A Nakano ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Transport ◽  
Cytoplasmic Domain ◽  
Temperature Sensitive ◽  
Membrane Glycoprotein ◽  
Permissive Temperature ◽  
Vesicle Budding ◽  
Terminal Domain ◽  
Transport Defect ◽  
Carboxy Terminal

Sec12p is a membrane glycoprotein required for the formation of a vesicular intermediate in protein transport from the endoplasmic reticulum to the Golgi apparatus in Saccharomyces cerevisiae. Comparison of the N-linked glycosylation of Sec12p, a Sec12p-invertase hybrid protein, and a derivative of Sec12p lacking 71 carboxy-terminal amino acids showed that Sec12p is a type II membrane protein. Analysis of two truncated forms of Sec12p and of a temperature-sensitive mutant indicated that the C-terminal domain of Sec12p is not essential for protein transport, whereas the integrity and membrane attachment of the cytoplasmic N-terminal domain are essential. Expression of a soluble cytoplasmic domain dramatically inhibited the growth of a sec12 temperature-sensitive strain by increasing the transport defect at a normally permissive temperature. This growth inhibition as well as the sec12 temperature-sensitive defect were suppressed by the overproduction of Sar1p, a small GTP-binding protein that participates in protein transport. Sar1p membrane association was enhanced by elevated levels of Sec12p. These results suggest that the cytoplasmic domain of Sec12p interacts with Sar1p and that the complex may function to promote vesicle formation.

The fission yeast bromodomain protein Bdf2 is required for growth of cells with circular chromosomes

2021 ◽  
Author(s):  
Misaki Yasuda ◽  
Ahmed G K Habib ◽  
Kanako Sugiura ◽  
Hossain Mohammad Shamim ◽  
Masaru Ueno
Keyword(s):  
Fission Yeast ◽  
High Temperatures ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
M Phase ◽  
Ts Mutant ◽  
Synthetically Lethal ◽  
Circular Chromosomes ◽  
Synthetic Growth

Abstract Circular chromosomes have frequently been observed in tumors of mesenchymal origin. In the fission yeast Schizosaccharomyces pombe, deletion of pot1+ results in rapid telomere loss, and the resulting survivors have circular chromosomes. Fission yeast has two bromodomains and extra-terminal (BET) proteins, Bdf1 and Bdf2; both are required for maintaining acetylated histones. Here, we found that bdf2, but not bdf1, was synthetically lethal with pot1. We also obtained a temperature-sensitive bdf2-ts mutant, which can grow at high temperatures but becomes camptothecin sensitive. This suggests that Bdf2 is defective at high temperatures. The cell cycle of the pot1 bdf2-ts mutant was delayed in the G2 and/or M phase at a semi-permissive temperature. Furthermore, a temperature-sensitive mutant of mst1, which encodes histone acetyltransferase, showed a synthetic growth defect with a pot1 disruptant at a semi-permissive temperature. Our results suggest that Bdf2 and Mst1 are required for the growth of cells with circular chromosomes.

Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins

1994 ◽  
Vol 107 (6) ◽  
pp. 1671-1678 ◽  
Author(s):  
G. Yu ◽  
J. Swiston ◽  
D. Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Adenylyl Cyclase ◽  
Normal Function ◽  
Temperature Sensitive ◽  
Related Protein ◽  
Adenylyl Cyclase Activity ◽  
Terminal Domain ◽  
Associated Proteins ◽  
Terminal Domains

We previously reported the identification of human CAP, a protein that is related to the Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylyl cyclase-associated CAP proteins. The two yeast CAP proteins have similar functions: the N-terminal domains are required for the normal function of adenylyl cyclase, while loss of the C-terminal domains result in morphological and nutritional defects that are unrelated to the cAMP pathways. We have amplified and cloned cDNAs from a human glioblastoma library that encode a second CAP-related protein, CAP2. The human CAP and CAP2 proteins are 64% identical. Expression of either human CAP or CAP2 in S. cerevisiae cap- strains suppresses phenotypes associated with deletion of the C-terminal domain of CAP, but does not restore hyper-activation of adenylyl cyclase by RAS2val19. Similarly, expression of either human CAP or CAP2 in S. pombe cap- strains suppresses the morphological and temperature-sensitive phenotypes associated with deletion of the C-terminal domain of CAP in this yeast. In addition, expression of human CAP, but not CAP2, suppresses the propensity to sporulate due to deletion of the N-terminal domain of CAP in S. pombe. This latter observation suggests that human CAP restores normal adenylyl cyclase activity in S. pombe cap- cells. Thus, functional properties of both N-terminal and C-terminal domains are conserved between the human and S. pombe CAP proteins.

scholarly journals Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly

1998 ◽  
Vol 143 (5) ◽  
pp. 1215-1225 ◽  
Author(s):  
José M. Barral ◽  
Christopher C. Bauer ◽  
Irving Ortiz ◽  
Henry F. Epstein
Keyword(s):  
Caenorhabditis Elegans ◽  
Central Region ◽  
Stop Codon ◽  
Common Mechanism ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Myosin Isoforms ◽  
Permissive Temperature ◽  
Terminal Domain ◽  
Abnormal Structure

The Caenorhabditis elegans unc-45 locus has been proposed to encode a protein machine for myosin assembly. The UNC-45 protein is predicted to contain an NH2-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p. CRO1 and She4p are fungal proteins required for the segregation of other molecules in budding, endocytosis, and septation. Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residues within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent translation of the COOH-terminal domain. Electron microscopy shows that thick filament accumulation in vivo is decreased by ∼50% in the CB286 ts mutant grown at the restrictive temperature. The thick filaments that assemble have abnormal structure. Immunofluorescence and immunoelectron microscopy show that myosins A and B are scrambled, in contrast to their assembly into distinct regions at the permissive temperature and in wild type. This abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortened lengths upon isolation. These results implicate the UNC-45 CRO1/She4p-like region in the assembly of myosin isoforms in C. elegans and suggest a possible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) protein family.

scholarly journals Faithful chromosome transmission requires Spt4p, a putative regulator of chromatin structure in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (6) ◽  
pp. 2838-2847 ◽  
Author(s):  
M A Basrai ◽  
J Kingsbury ◽  
D Koshland ◽  
F Spencer ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Instability ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Chromosome Fragment ◽  
Dna Mutations ◽  
Chromosome Transmission ◽  
Biochemical Assays

A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae SPT4 gene. The s138 mutation is a missense mutation in the second of four conserved cysteine residues positioned similarly to those of zinc finger proteins, and we henceforth refer to the mutation of spt4-138. Both spt4-138 and spt4 delta strains missegregate a chromosome fragment at the permissive temperature, are temperature sensitive for growth at 37 degrees C, and upon a shift to the nonpermissive temperature show an accumulation of large budded cells, each with a nucleus. Previous studies suggest that Spt4p functions in a complex with Spt5p and Spt6p, and we determined that spt6-140 also causes missegregation of a chromosome fragment. Double mutants carrying spt4 delta 2::HIS3 and kinetochore mutation ndc10-42 or ctf13-30 show a synthetic conditional phenotype. Both spt4-138 and spt4 delta strains exhibit synergistic chromosome instability in combination with CEN DNA mutations and show in vitro defects in microtubule binding to minichromosomes. These results indicate that Spt4p plays a role in chromosome segregation. The results of in vivo genetic interactions with mutations in kinetochore proteins and CEN DNA and of in vitro biochemical assays suggest that Spt4p is important for kinetochore function.

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