Sit4p Protein Phosphatase Is Required for Sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis Zymocin

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1479-1489
Author(s):  
Daniel Jablonowski ◽  
Andrew R Butler ◽  
Lars Fichtner ◽  
Donald Gardiner ◽  
Raffael Schaffrath ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Protein Phosphatase ◽  
Kluyveromyces Lactis ◽  
The Other ◽  
G1 Phase ◽  
Elongator Complex ◽  
Intracellular Target ◽  
Related Proteins

Abstract We have identified two Saccharomyces cerevisiae genes that, in high copy, confer resistance to Kluyveromyces lactis zymocin, an inhibitor that blocks cells in the G1 phase of the cell cycle prior to budding and DNA replication. One gene (GRX3) encodes a glutaredoxin and is likely to act at the level of zymocin entry into sensitive cells, while the other encodes Sap155p, one of a family of four related proteins that function positively and interdependently with the Sit4p protein phosphatase. Increased SAP155 dosage protects cells by influencing the sensitivity of the intracellular target and is unique among the four SAP genes in conferring zymocin resistance in high copy, but is antagonized by high-copy SAP185 or SAP190. Since cells lacking SIT4 or deleted for both SAP185 and SAP190 are also zymocin resistant, our data support a model whereby high-copy SAP155 promotes resistance by competition with the endogenous levels of SAP185 and SAP190 expression. Zymocin sensitivity therefore requires a Sap185p/Sap190p-dependent function of Sit4p protein phosphatase. Mutations affecting the RNA polymerase II Elongator complex also confer K. lactis zymocin resistance. Since sit4Δ and SAP-deficient strains share in common several other phenotypes associated with Elongator mutants, Elongator function may be a Sit4p-dependent process.

scholarly journals Absence of step changes in activity of certain enzymes during the cell cycle of budding and fission yeasts in synchronous cultures

1983 ◽  
Vol 61 (1) ◽  
pp. 339-349
Author(s):  
J. Creanor ◽  
S.G. Elliott ◽  
Y.C. Bisset ◽  
J.M. Mitchison
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Acid Phosphatase ◽  
Kluyveromyces Lactis ◽  
The Other ◽  
Linear Pattern ◽  
Synchronous Cultures ◽  
Asynchronous Control ◽  
Alpha Glucosidase ◽  
Beta Galactosidase

Synchronous cultures prepared by selection from an elutriating rotor were used to measure activity changes during the cell cycle of the following enzymes: acid phosphatase in Schizosaccharomyces pombe and Saccharomyces cerevisiae, alpha-glucosidase in S. cerevisiae and beta-galactosidase in Kluyveromyces lactis. There was no sign of step rises in activity in acid phosphatase but there were indications in S. cerevisiae of the linear pattern with rate doublings once per cycle that had been found previously in S. pombe. There was also no sign of step rises in the other two enzymes, in contrast to earlier results using different techniques. Asynchronous control cultures showed little or no perturbations after the first hour.

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

scholarly journals Recombination of Ty elements in yeast can be induced by a double-strand break.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 67-77 ◽  
Author(s):  
A Parket ◽  
O Inbar ◽  
M Kupiec
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Strand Break ◽  
Double Strand Break ◽  
The Other ◽  
Direct Repeats ◽  
Yeast Saccharomyces Cerevisiae ◽  
Low Frequencies ◽  
Ty Elements ◽  
Main Family

Abstract The Ty retrotransposons are the main family of dispersed repeated sequences in the yeast Saccharomyces cerevisiae. These elements are flanked by a pair of long terminal direct repeats (LTRs). Previous experiments have shown that Ty elements recombine at low frequencies, despite the fact that they are present in 30 copies per genome. This frequency is not highly increased by treatments that cause DNA damage, such as UV irradiation. In this study, we show that it is possible to increase the recombination level of a genetically marked Ty by creating a double-strand break in it. This break is repaired by two competing mechanisms: one of them leaves a single LTR in place of the Ty, and the other is a gene conversion event in which the marked Ty is replaced by an ectopically located one. In a strain in which the marked Ty has only one LTR, the double-strand break is repaired by conversion. We have also measured the efficiency of repair and monitored the progression of the cells through the cell-cycle. We found that in the presence of a double-strand break in the marked Ty, a proportion of the cells is unable to resume growth.

scholarly journals DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4675-4684 ◽  
Author(s):  
F R Cross
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Critical Size ◽  
Size Control ◽  
Mutant Gene ◽  
G1 Phase ◽  
Wild Type ◽  
Alpha Factor ◽  
Multiple Copies ◽  
Kinetics Of

The mating pheromone alpha-factor arrests Saccharomyces cerevisiae MATa cells in the G1 phase of the cell cycle. Size control is also exerted in G1, since cells do not exit G1 until they have attained a critical size. A dominant mutation (DAF1-1) which causes both alpha-factor resistance and small cell size (volume about 0.6-fold that of the wild type) has been isolated and characterized genetically and by molecular cloning. Several alpha-factor-induced mRNAs were induced equivalently in daf1+ and DAF1-1 cells. The DAF1-1 mutation consisted of a termination codon two-thirds of the way through the daf1+ coding sequence. A chromosomal deletion of DAF1 produced by gene transplacement increased cell volume about 1.5-fold; thus, DAF1-1 may be a hyperactive or deregulated allele of a nonessential gene involved in G1 size control. Multiple copies of DAF1-1 also greatly reduced the duration of the G1 phase of the cell cycle.

scholarly journals Dynamic Localization of Protein Phosphatase Type 1 in the Mitotic Cell Cycle of Saccharomyces cerevisiae

2000 ◽  
Vol 149 (1) ◽  
pp. 125-140 ◽  
Author(s):  
Andrew Bloecher ◽  
Kelly Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Phosphatase ◽  
Essential Gene ◽  
Mitotic Cell ◽  
Type I ◽  
Mitotic Cell Cycle ◽  
Dependent Manner ◽  
Bud Neck ◽  
Protein Phosphatase Type

Protein phosphatase type I (PP1), encoded by the single essential gene GLC7 in Saccharomyces cerevisiae, functions in diverse cellular processes. To identify in vivo subcellular location(s) where these processes take place, we used a functional green fluorescent protein (GFP)–Glc7p fusion protein. Time-lapse fluorescence microscopy revealed GFP–Glc7p localizes predominantly in the nucleus throughout the mitotic cell cycle, with the highest concentrations in the nucleolus. GFP–Glc7p was also observed in a ring at the bud neck, which was dependent upon functional septins. Supporting a role for Glc7p in bud site selection, a glc7-129 mutant displayed a random budding pattern. In α-factor treated cells, GFP–Glc7p was located at the base of mating projections, again in a septin-dependent manner. At the start of anaphase, GFP–Glc7p accumulated at the spindle pole bodies and remained there until cytokinesis. After anaphase, GFP–Glc7p became concentrated in a ring that colocalized with the actomyosin ring. A GFP–Glc7-129 fusion was defective in localizing to the bud neck and SPBs. Together, these results identify sites of Glc7p function and suggest Glc7p activity is regulated through dynamic changes in its location.

X-ray microanalysis of HeLa S3 cells. II. Analysis of elemental levels during the cell cycle

1983 ◽  
Vol 62 (1) ◽  
pp. 339-350
Author(s):  
A. Warley ◽  
J. Stephen ◽  
A. Hockaday ◽  
T.C. Appleton
Keyword(s):  
Cell Cycle ◽  
Monovalent Cation ◽  
The Other ◽  
G1 Phase ◽  
Sodium Potassium ◽  
X Ray ◽  
Cation Concentration ◽  
Cell Compartments ◽  
Hela S3 ◽  
Hela S3 Cells

HeLa S3 cells were synchronized using hydroxyurea. Cryoultramicrotomy and X-ray microanalysis were used to study changes occurring in concentrations of elements during the cell cycle of the synchronized cells. Three subcellular compartments were studied: cytoplasm, nucleus and nucleolus. Potassium concentrations showed little fluctuation in all of the cell compartments during the cell cycle. Sodium concentrations increased during S. and M phases, returning to lower levels in the G1 phase. Chlorine concentrations were highest during the S and G2 phases. At all stages of the cell cycle respective concentrations of potassium, sodium, sulphur and chlorine were similar in the cytoplasm and nucleus. Concentrations of phosphorus increased in the nucleus during S, G2 and M, and also showed fluctuations in the nucleolus during the cycle; these were not seen in the cytoplasm. In S, M and M/G1 sodium concentrations were highest in the nucleolus compared with the other compartments. In the cytoplasm these changes resulted in an increase in total monovalent cation concentration (i.e. sodium + potassium) during S, G2 and M, which returned to base levels after mitosis. This increase in monovalent cation concentration is due almost entirely to the increase in sodium, with little change occurring in the concentration of potassium.

scholarly journals Structure-function relationships of the yeast cyclin-dependent kinase Pho85.

1995 ◽  
Vol 15 (10) ◽  
pp. 5482-5491 ◽  
Author(s):  
R C Santos ◽  
N C Waters ◽  
C L Creasy ◽  
L W Bergman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Structure Function ◽  
Cell Cycle Progression ◽  
Substrate Recognition ◽  
The Other ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Induced Mutagenesis

The PHO85 gene of Saccharomyces cerevisiae encodes a cyclin-dependent kinase involved in both transcriptional regulation and cell cycle progression. Although a great deal is known concerning the structure, function, and regulation of the highly homologous Cdc28 protein kinase, little is known concerning these relationships in regard to Pho85. In this study, we constructed a series of Pho85-Cdc28 chimeras to map the region(s) of the Pho85 molecule that is critical for function of Pho85 in repression of acid phosphatase (PHO5) expression. Using a combination of site-directed and ethyl methanesulfonate-induced mutagenesis, we have identified numerous residues critical for either activation of the Pho85 kinase, interaction of Pho85 with the cyclin-like molecule Pho80, or substrate recognition. Finally, analysis of mutations analogous to those previously identified in either Cdc28 or cdc2 of Schizosaccharomyces pombe suggested that the inhibition of Pho85-Pho80 activity in mechanistically different from that seen in the other cyclin-dependent kinases.

scholarly journals Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A.

1997 ◽  
Vol 17 (6) ◽  
pp. 3242-3253 ◽  
Author(s):  
Y Shu ◽  
H Yang ◽  
E Hallberg ◽  
R Hallberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
High Temperatures ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Molecular Genetic Analysis ◽  
Regulatory Subunit ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Phosphatase 2A

The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation.

scholarly journals The Yeast Elongator Histone Acetylase Requires Sit4-dependent Dephosphorylation for Toxin-Target Capacity

2004 ◽  
Vol 15 (3) ◽  
pp. 1459-1469 ◽  
Author(s):  
Daniel Jablonowski ◽  
Lars Fichtner ◽  
Michael J.R. Stark ◽  
Raffael Schaffrath
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Kluyveromyces Lactis ◽  
Toxin Complex ◽  
Cell Cycle Block ◽  
Resistant Cells

Kluyveromyces lactis zymocin, a heterotrimeric toxin complex, imposes a G1 cell cycle block on Saccharomyces cerevisiae that requires the toxin-target (TOT) function of holo-Elongator, a six-subunit histone acetylase. Here, we demonstrate that Elongator is a phospho-complex. Phosphorylation of its largest subunit Tot1 (Elp1) is supported by Kti11, an Elongator-interactor essential for zymocin action. Tot1 dephosphorylation depends on the Sit4 phosphatase and its associators Sap185 and Sap190. Zymocin-resistant cells lacking or overproducing Elongator-associator Tot4 (Kti12), respectively, abolish or intensify Tot1 phosphorylation. Excess Sit4·Sap190 antagonizes the latter scenario to reinstate zymocin sensitivity in multicopy TOT4 cells, suggesting physical competition between Sit4 and Tot4. Consistently, Sit4 and Tot4 mutually oppose Tot1 de-/phosphorylation, which is dispensable for integrity of holo-Elongator but crucial for the TOT-dependent G1 block by zymocin. Moreover, Sit4, Tot4, and Tot1 cofractionate, Sit4 is nucleocytoplasmically localized, and sit4Δ-nuclei retain Tot4. Together with the findings that sit4Δ and totΔ cells phenocopy protection against zymocin and the ceramide-induced G1 block, Sit4 is functionally linked to Elongator in cell cycle events targetable by antizymotics.

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