Regulation of p105weel and p34cdc2 during meiosis in Schizosaccharomyces pombe

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1088-1096 ◽  
Author(s):  
Maleki Daya-Makin ◽  
Philippe Szankasi ◽  
Liren Tang ◽  
Diana MacRae ◽  
Steven L. Pelech
Keyword(s):  
Protein Kinase ◽  
Schizosaccharomyces Pombe ◽  
Thermal Inactivation ◽  
State Level ◽  
Fold Increase ◽  
Histone H1 ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Nutritional Conditions ◽  
Tyrosyl Phosphorylation

Temperature-sensitive pat1 mutants of the fission yeast Schizosaccharomyces pombe can be induced to undergo meiosis at the restrictive temperature, irrespective of the mat1 configuration and the nutritional conditions. Using a combination of exit from stationary phase and thermal inactivation of the 52-kilodalton protein kinase that is encoded by the pat1 (also called ran1) gene, highly synchronous meiotic cultures were obtained. Synthesis and tyrosyl phosphorylation of p34cdc2 was evident during meiotic G1 and S phases. During this period there was increased expression of p105wee1, a protein kinase implicated in the tyrosyl phosphorylation of p34cdc2. Following a relatively brief G2 period, during which a reduction in the steady-state level of p105wee1 occurred, there was an approximately 19-fold increase in the histone H1 phosphotransferase activity of p34cdc2. Only a single peak of histone H1 kinase activation was observed, which implies that unlike meiosis in amphibians and echinoderms, p34cdc2 is functional only during one of the meiotic divisions in S. pombe, presumably meiosis II. Stimulation of the kinase activity of p34cdc2 was associated with its tyrosyl dephosphorylation. This is analogous to mitotic M phase and suggests parallels in the mechanism of activation of p34cdc2 during mitosis and one of the meiotic divisions in S. pombe.Key words: wee1, cdc2, ran1, cell cycle, meiosis.

scholarly journals The Schizosaccharomyces pombe dim1+ Gene Interacts with the Anaphase-Promoting Complex or Cyclosome (APC/C) Component lid1+ and Is Required for APC/C Function

1999 ◽  
Vol 19 (4) ◽  
pp. 2535-2546 ◽  
Author(s):  
Lynne D. Berry ◽  
Anna Feoktistova ◽  
Melanie D. Wright ◽  
Kathleen L. Gould
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Chromosome Segregation ◽  
State Level ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Anaphase Promoting Complex ◽  
Synthetic Lethal ◽  
C Complex ◽  
Mutant Cells

ABSTRACT The Schizosaccharomyces pombe dim1 + gene is required for entry into mitosis and for chromosome segregation during mitosis. To further understand dim1p function, we undertook a synthetic lethal screen with the temperature-sensitive dim1-35 mutant and isolated lid (for lethal in dim1-35) mutants. Here, we describe the temperature-sensitive lid1-6mutant. At the restrictive temperature of 36°C, lid1-6mutant cells arrest with a “cut” phenotype similar to that ofcut4 and cut9 mutants. An epitope-tagged version of lid1p is a component of a multiprotein ∼20S complex; the presence of lid1p in this complex depends upon functionalcut9 +. lid1p-myc coimmunoprecipitates with several other proteins, including cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity oflid1 +. Further, lid1 +function is required for the multiubiquitination of cut2p, an anaphase-promoting complex or cyclosome (APC/C) target. Thus, lid1p is a component of the S. pombe APC/C. In dim1mutants, the abundances of lid1p and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished. These data suggest that at least one role of dim1p is to maintain or establish the steady-state level of the APC/C.

scholarly journals Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

1993 ◽  
Vol 13 (5) ◽  
pp. 2870-2881 ◽  
Author(s):  
L C Robinson ◽  
M M Menold ◽  
S Garrett ◽  
M R Culbertson
Keyword(s):  
Protein Kinase ◽  
Eukaryotic Cell ◽  
Casein Kinase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Restrictive Temperature ◽  
Loss Of Function ◽  
Casein Kinase I ◽  
Yeast Saccharomyces Cerevisiae

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

Change in the rate of CO2 production in synchronous cultures of the fission yeast Schizosaccharomyces pombe: a periodic cell cycle event that persists after the DNA-division cycle has been blocked

1986 ◽  
Vol 86 (1) ◽  
pp. 191-206
Author(s):  
B. Novak ◽  
J.M. Mitchison
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Rate Change ◽  
Co2 Production ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Normal Cycle ◽  
Periodic Cell ◽  
Linear Pattern ◽  
Synchronous Cultures ◽  
Oscillatory Pattern

CO2 production has been followed by manometry in synchronous and asynchronous cultures of Schizosaccharomyces pombe prepared by elutriation from the same initial culture. The rate of production follows a linear pattern in synchronous cultures with a rate change once per cycle at the time of cell division. This pattern is most clearly shown in oscillations of the difference between values of the second differential (acceleration) for the synchronous and asynchronous cultures. The association between the rate change and the time of division is maintained during growth speeded up in rich medium and slowed down in poor medium and at lower temperature. It is also maintained after a shift-up in temperature. Results with wee mutants suggest that the association is with the S period rather than division itself. The rate and acceleration of CO2 production are approximately proportional to cell size (protein content) in asynchronous cultures. When synchronous cultures of the temperature-sensitive mutants cdc2.33 and cdc2.33 wee1.6 are shifted up to the restrictive temperature, the DNA-division cycle is blocked. The oscillatory pattern of CO2 production, however, continues for one to two cycles until the acceleration reaches a constant value, after which the oscillations are undetectable. This point is reached later in the double mutant and there is a phase difference in the oscillations compared to those in the single mutant. With both blocked mutants the ‘free-running’ oscillations are about 15% shorter than the normal cycle time. There are well-known examples of such oscillations in eggs but they are rare in growing systems.

scholarly journals Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

1982 ◽  
Vol 2 (2) ◽  
pp. 199-206 ◽  
Author(s):  
T D Gilmore ◽  
K Radke ◽  
G S Martin
Keyword(s):  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Defective Mutant ◽  
Sarcoma Virus

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.

scholarly journals The Schizosaccharomyces pombe rad60 Gene Is Essential for Repairing Double-Strand DNA Breaks Spontaneously Occurring during Replication and Induced by DNA-Damaging Agents

2002 ◽  
Vol 22 (10) ◽  
pp. 3537-3548 ◽  
Author(s):  
Takashi Morishita ◽  
Yasuhiro Tsutsui ◽  
Hiroshi Iwasaki ◽  
Hideo Shinagawa
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Strand Break ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Multicopy Plasmid ◽  
Dna Breaks ◽  
Γ Irradiation ◽  
Synthetic Lethal ◽  
Double Strand Dna Breaks ◽  
Dna Double Strand Break

ABSTRACT To identify novel genes involved in DNA double-strand break (DSB) repair, we previously isolated Schizosaccharomyces pombe mutants which are hypersensitive to methyl methanesulfonate (MMS) and synthetic lethals with rad2. This study characterizes one of these mutants, rad60-1. The gene that complements the MMS sensitivity of this mutant was cloned and designated rad60. rad60 encodes a protein with 406 amino acids which has the conserved ubiquitin-2 motif found in ubiquitin family proteins. rad60-1 is hypersensitive to UV and γ rays, epistatic to rhp51, and defective in the repair of DSBs caused by γ-irradiation. The rad60-1 mutant is also temperature sensitive for growth. At the restrictive temperature (37°C), rad60-1 cells grow for several divisions and then arrest with 2C DNA content; the arrested cells accumulate DSBs and have a diffuse and often aberrantly shaped nuclear chromosomal domain. The rad60-1 mutant is a synthetic lethal with rad18-X, and expression of wild-type rad60 from a multicopy plasmid partially suppresses the MMS sensitivity of rad18-X cells. rad18 encodes a conserved protein of the structural maintenance of chromosomes (SMC) family (A. R. Lehmann, M. Walicka, D. J. Griffiths, J. M. Murray, F. Z. Watts, S. McCready, and A. M. Carr, Mol. Cell. Biol. 15:7067-7080, 1995). These results suggest that S. pombe Rad60 is required to repair DSBs, which accumulate during replication, by recombination between sister chromatids. Rad60 may perform this function in concert with the SMC protein Rad18.

scholarly journals Myristic acid auxotrophy caused by mutation of S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

1991 ◽  
Vol 113 (6) ◽  
pp. 1313-1330 ◽  
Author(s):  
R J Duronio ◽  
D A Rudnick ◽  
R L Johnson ◽  
D R Johnson ◽  
J I Gordon
Keyword(s):  
Myristic Acid ◽  
Fatty Acid Synthetase ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Myristoyl Coa

The S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth. NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy. The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36 degrees C) is rescued with exogenous C14:0. Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24 degrees C). Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24 degrees C. Complementation of growth at 36 degrees C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene. The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451----Asp substitution. Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis. In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent Km for myristoyl-CoA at 36 degrees C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency. Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.

scholarly journals NHP6A and NHP6B, which encode HMG1-like proteins, are candidates for downstream components of the yeast SLT2 mitogen-activated protein kinase pathway.

1994 ◽  
Vol 14 (4) ◽  
pp. 2391-2403 ◽  
Author(s):  
C Costigan ◽  
D Kolodrubetz ◽  
M Snyder
Keyword(s):  
Protein Kinase ◽  
Synthetic Lethality ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitogen Activated Protein ◽  
Multiple Copies ◽  
Delta Cells

The yeast SLK1 (BCK1) gene encodes a mitogen-activated protein kinase (MAPK) activator protein which functions upstream in a protein kinase cascade that converges on the MAPK Slt2p (Mpk1p). Dominant alleles of SLK1 have been shown to bypass the conditional lethality of a protein kinase C mutation, pkc1-delta, suggesting that Pkc1p may regulate Slk1p function. Slk1p has an important role in morphogenesis and growth control, and deletions of the SLK1 gene are lethal in a spa2-delta mutant background. To search for genes that interact with the SLK1-SLT2 pathway, a synthetic lethal suppression screen was carried out. Genes which in multiple copies suppress the synthetic lethality of slk1-1 spa2-delta were identified, and one, the NHP6A gene, has been extensively characterized. The NHP6A gene and the closely related NHP6B gene were shown previously to encode HMG1-like chromatin-associated proteins. We demonstrate here that these genes are functionally redundant and that multiple copies of either NHP6A or NHP6B suppress slk1-delta and slt2-delta. Strains from which both NHP6 genes were deleted (nhp6-delta mutants) share many phenotypes with pkc1-delta, slk1-delta, and slt2-delta mutants. nhp6-delta cells display a temperature-sensitive growth defect that is rescued by the addition of 1 M sorbitol to the medium, and they are sensitive to starvation. nhp6-delta strains also exhibit a variety of morphological and cytoskeletal defects. At the restrictive temperature for growth, nhp6-delta mutant cells contain elongated buds and enlarged necks. Many cells have patches of chitin staining on their cell surfaces, and chitin deposition is enhanced at the necks of budded cells. nhp6-delta cells display a defect in actin polarity and often accumulate large actin chunks. Genetic and phenotypic analysis indicates that NHP6A and NHP6B function downstream of SLT2. Our results indicate that the Slt2p MAPK pathway in Saccharomyces cerevisiae may mediate its function in cell growth and morphogenesis, at least in part, through high-mobility group proteins.

scholarly journals A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.

1988 ◽  
Vol 106 (4) ◽  
pp. 1171-1183 ◽  
Author(s):  
T Hirano ◽  
Y Hiraoka ◽  
M Yanagida
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Gradient Centrifugation ◽  
Metaphase Plate ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
In The Wild ◽  
Spindle Elongation ◽  
Mutant Cells

A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.

The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex

1989 ◽  
Vol 9 (5) ◽  
pp. 2034-2041
Author(s):  
J A Hadwiger ◽  
C Wittenberg ◽  
M D Mendenhall ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Schizosaccharomyces Pombe ◽  
Temperature Sensitive ◽  
G1 Arrest ◽  
Genes Encoding ◽  
A Subunit ◽  
Identity Disruption ◽  
High Degree ◽  
High Copy Plasmid

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for cell cycle initiation. In an attempt to identify genes encoding proteins that interact with the Cdc28 protein kinase, high-copy plasmid suppressors of a temperature-sensitive cdc28 mutation were isolated. One such suppressor, CKS1, was found to encode an 18-kilodalton protein that shared a high degree of homology with the suc1+ protein (p13) of Schizosaccharomyces pombe (67% amino acid sequence identity). Disruption of the chromosomal CKS1 gene conferred a G1 arrest phenotype similar to that of cdc28 mutants. The presence of the 18-kilodalton Cks1 protein in yeast lysates was demonstrated by using Cks-1 specific antiserum. Furthermore, the Cks1 protein was shown to be physically associated with active forms of the Cdc28 protein kinase. These data suggest that Cks1 is an essential component of the Cdc28 protein kinase complex.

An Essential Function of a Phosphoinositide-Specific Phospholipase C Is Relieved by Inhibition of a Cyclin-Dependent Protein Kinase in the Yeast Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 33-47 ◽  
Author(s):  
Jeffrey S Flick ◽  
Jeremy Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Complete Medium ◽  
Restrictive Temperature ◽  
Dependent Protein Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dependent Protein

Abstract The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the δ isoform of mammalian phosphoinositide-specific phospholipase C (PI-PLC). We found that two genes (SPL1 and SPL2), when overexpressed, can bypass the temperature-sensitive growth defect of a plc1Δ cell. SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk). In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80Δ) mutation and growth on low-phosphate medium, also permitted growth of plc1Δ cells at the restrictive temperature. Suppression of the temperature sensitivity of plc1Δ cells by pho80Δ does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk. The second suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors. Both pho81Δ and spl2Δ show a synthetic phenotype in combination with plc1Δ. Unlike single mutants, plc1Δ pho81Δ and plc1Δ spl2Δ double mutants were unable to grow on synthetic complete medium, but were able to grow on rich medium.

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