scholarly journals Bypassing anaphase by fission yeast cut9 mutation: requirement of cut9+ to initiate anaphase.

1994 ◽  
Vol 127 (6) ◽  
pp. 1655-1670 ◽  
Author(s):  
I Samejima ◽  
M Yanagida
Keyword(s):  
Amino Acid ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Temperature Sensitive ◽  
Cytoplasmic Microtubule ◽  
Tetratricopeptide Repeats ◽  
Late Anaphase ◽  
Amino Acid Repeats ◽  
Cold Sensitive ◽  
Essential Function

A novel anaphase block phenotype was found in fission yeast temperature-sensitive cut9 mutants. Cells enter mitosis with chromosome condensation and short spindle formation, then block anaphase, but continue to progress into postanaphase events such as degradation of the spindle, reformation of the postanaphase cytoplasmic microtubule arrays, septation, and cytokinesis. The cut9 mutants are defective in the onset of anaphase and possibly in the restraint of postanaphase events until the completion of anaphase. The cut9+ gene encodes a 78-kD protein containing the 10 34-amino acid repeats, tetratricopeptide repeats (TPR), and similar to budding yeast Cdc16. It is essential for viability, and the mutation sites reside in the TPR. The three genes, namely, nuc2+, scn1+, and scn2+, genetically interact with cut9+. The nuc2+ and cut9+ genes share an essential function to initiate anaphase. The cold-sensitive scn1 and scn2 mutations, defective in late anaphase, can suppress the ts phenotype of cut9.

The novel human protein serine/threonine phosphatase 6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, which are involved in cell cycle regulation

1996 ◽  
Vol 109 (12) ◽  
pp. 2865-2874 ◽  
Author(s):  
H. Bastians ◽  
H. Ponstingl
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Shape Control ◽  
Cell Cycle Regulation ◽  
Budding Yeast ◽  
Mitotic Checkpoint ◽  
Human Protein ◽  
Predicted Amino Acid Sequence ◽  
Temperature Sensitive ◽  
Cycle Regulation

We identified a novel human protein serine/threonine phosphatase cDNA, designated protein phosphatase 6 (PP6) by using a homology-based polymerase chain reaction. The predicted amino acid sequence indicates a 35 kDa protein showing high homology to other protein phosphatases including human PP2A (57%), human PP4 (59%), rat PPV (98%), Drosophila PPV (74%), Schizosaccharomyces pombe ppe1 (68%) and Saccharomyces cerevisiae Sit4p (61%). In human cells, three forms of PP6 mRNA were found with highest levels of expression in testis, heart and skeletal muscle. The PP6 protein was detected in lysates of human heart muscle and in bull testis. Complementation studies using a temperature sensitive mutant strain of S. cerevisiae SIT4, which is required for the G1 to S transition of the cell cycle, showed that PP6 can rescue the mutant growth arrest. In addition, a loss of function mutant of S. pombe ppe1, described as a gene interacting with the pim1/spi1 mitotic checkpoint and involved in cell shape control, can be complemented by expression of human PP6. These data indicate that human PP6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, implying a function of PP6 in cell cycle regulation.

scholarly journals Contribution of Alanine-76 and Serine Phosphorylation inα-Synuclein Membrane Association and Aggregation in Yeasts

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Michael Fiske ◽  
Stephanie Valtierra ◽  
Keith Solvang ◽  
Michael Zorniak ◽  
Michael White ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Fission Yeast ◽  
Dopaminergic Neurons ◽  
Budding Yeast ◽  
Serine Phosphorylation ◽  
Membrane Association ◽  
Midbrain Dopaminergic Neurons ◽  
Substitution Mutations

In Parkinson's disease (PD), misfolded and aggregatedα-synuclein protein accumulates in degenerating midbrain dopaminergic neurons. The amino acid alanine-76 inα-synuclein and phosphorylation at serine-87 and serine-129 are thought to regulate its aggregation and toxicity. However, their exact contributions toα-synuclein membrane association are less clear. We found thatα-synuclein is indeed phosphorylated in fission yeast and budding yeast, the two models that we employed for assessingα-synuclein aggregation and membrane association properties, respectively. Surprisingly, blocking serine phosphorylation (S87A, S129A, and S87A/S129A) or mimicking it (S87D, S129D) alteredα-synuclein aggregation in fission yeast. Either blocking or mimicking this phosphorylation increased endomembrane association in fission yeast, but only mimicking it decreased plasma membrane association in budding yeast. Polar substitution mutations of alanine-76 (A76E and A76R) decreasedα-synuclein membrane association in budding yeast and decreased aggregation in fission yeast. These yeast studies extend our understanding of serine phosphorylation and alanine-76 contributions toα-synuclein aggregation and are the first to detail their impact onα-synuclein's plasma membrane and endomembrane association.

scholarly journals Identification of a conserved F-box protein 6 interactor essential for endocytosis and cytokinesis in fission yeast

2009 ◽  
Vol 420 (2) ◽  
pp. 169-180 ◽  
Author(s):  
Isabelle Jourdain ◽  
Nathalie Spielewoy ◽  
James Thompson ◽  
Susheela Dhut ◽  
John R. Yates ◽  
...  
Keyword(s):  
Cell Division ◽  
Fission Yeast ◽  
Protein Identification ◽  
Budding Yeast ◽  
Affinity Purification ◽  
Elongation Factor ◽  
Temperature Sensitive ◽  
Amino Acid Residues ◽  
Lipid Kinase ◽  
F Box Protein

The F-box domain is a degenerated motif consisting of ∼40 amino acid residues that specifically bind Skp1, a core component of the SCF (Skp1-Cdc53/Cullin 1-F-box protein) ubiquitin ligase. Recent work, mainly performed in budding yeast, indicates that certain F-box proteins form non-SCF complexes together with Skp1 in the absence of cullins and play various roles in cell cycle and signalling pathways. However, it is not established whether these non-SCF complexes are unique to budding yeast or common in other eukaryotes. In the present paper, using TAP (tandem affinity purification) coupled to MudPIT (Multidimensional Protein Identification Technology) analysis, we have identified a novel conserved protein, Sip1, in fission yeast, as an interacting partner of an essential F-box protein Pof6. Sip1 is a large HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor)-repeats containing protein (217 kDa) and forms a complex with Pof6 and Skp1. This complex does not contain cullins, indicating that it is a novel non-SCF complex. Like Pof6 and Skp1, Sip1 is essential for cell viability and temperature-sensitive sip1 mutants display cell division arrest as binucleate cells with septa. Sip1 localizes to the nucleus and dynamic cytoplasmic dots, which are shown in the present study to be endocytic vesicles. Consistent with this, sip1 mutants are defective in endocytosis. Furthermore, towards the end of cytokinesis, constriction of the actomyosin ring and dissociation of type II myosin and septum materials are substantially delayed in the absence of functional Sip1. These results indicate that the conserved Sip1 protein comprises a novel non-SCF F-box complex that plays an essential role in endocytosis, cytokinesis and cell division.

scholarly journals A Fourth Component of the Fission Yeast γ-Tubulin Complex, Alp16, Is Required for Cytoplasmic Microtubule Integrity and Becomes Indispensable When γ-Tubulin Function Is Compromised

2002 ◽  
Vol 13 (7) ◽  
pp. 2360-2373 ◽  
Author(s):  
Akiko Fujita ◽  
Leah Vardy ◽  
Miguel Angel Garcia ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Microtubule ◽  
Multicopy Suppressor ◽  
Large Complex ◽  
Synthetically Lethal ◽  
And Function

γ-Tubulin functions as a multiprotein complex, called the γ-tubulin complex (γ-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved γ-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16 + , as a multicopy suppressor of temperature-sensitive alp6-719mutants. alp16 + encodes a 759-amino-acid protein with two conserved regions found in all other members of γ-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16 + is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225,alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with γ-tubulin and cosediments with the γ-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the γ-TuC between yeast and higher eukaryotes.

scholarly journals Identification of cut8+ and cek1+, a novel protein kinase gene, which complement a fission yeast mutation that blocks anaphase.

1994 ◽  
Vol 14 (9) ◽  
pp. 6361-6371 ◽  
Author(s):  
I Samejima ◽  
M Yanagida
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Fission Yeast ◽  
Gene Product ◽  
Budding Yeast ◽  
Essential Gene ◽  
Restrictive Temperature ◽  
Kinase Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Novel Protein

The fission yeast Schizosaccharomyces pombe [corrected] temperature sensitivity cut8-563 mutation causes chromosome overcondensation and short spindle formation in the absence of sister chromatid separation. The cut8-563 mutation allows cytokinesis before the completion of anaphase, thus producing cells with a cut phenotype. The cut8+ gene product may be required for normal progression of anaphase. Diploidization occurs at the restrictive temperature, and 60 to 70% of the cells surviving after two generations are diploid. These phenotypes are reminiscent of those of budding yeast (Saccharomyces cerevisiae) ctf13 and ctf14 (ndc10) mutations. The cut8+ gene, isolated by complementation of the mutant, predicts a 262-amino-acid protein; the amino and carboxy domains are hydrophilic, while the central domain contains several hydrophobic stretches. It has a weak overall similarity to the budding yeast DBF8 gene product. DBF8 is an essential gene whose mutations result in delay in mitotic progression and chromosome instability. Anti-cut8 antibodies detect a 33-kDa polypeptide. Two multicopy suppressor genes for cut8-563 are identified. They are the cut1+ gene essential for nuclear division, and a new gene (designated cek1+) which encodes a novel protein kinase. The cek1+ gene product is unusually large (1,309 amino acids) and has a 112-amino-acid additional sequence in the kinase domain. The cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein.

scholarly journals Isolation and characterization of the fission yeast protein phosphatase gene ppe1+ involved in cell shape control and mitosis.

1993 ◽  
Vol 4 (3) ◽  
pp. 303-313 ◽  
Author(s):  
M Shimanuki ◽  
N Kinoshita ◽  
H Ohkura ◽  
T Yoshida ◽  
T Toda ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Shape Control ◽  
Budding Yeast ◽  
Suppressor Gene ◽  
Predicted Amino Acid Sequence ◽  
Permissive Temperature ◽  
Multicopy Suppressor ◽  
Isolation And Characterization ◽  
Cold Sensitive ◽  
Cell Shape Control

We isolated a fission yeast putative protein serine/threonine phosphatase gene designated ppe1+ by hybridization. The predicted amino acid sequence is similar to those of the fission yeast ppa2 (53% identity) and dis2 (39%) phosphatases, and highly similar to those of the budding yeast SIT4 (72%), Drosophila PPV (68%) and rabbit PPX (61%) phosphatases. Antibodies against ppe1 protein identified a 37-kd polypeptide in fission yeast. A gene disruption (designated delta ppe1) caused cold-sensitive lethality and short, pear-shaped cells. These phenotypes were fully suppressed by a plasmid carrying ppe1+. Three classes of multicopy suppressor genes for delta ppe1 were identified as follows: 1) ppa1+ and ppa2+ encoding type 2A-like phosphatases, 2) mitotically essential dis3+ similar to the budding yeast SSD1/SRK1, a suppressor for sit4, and 3) pck1+ coding for a protein kinase C-like kinase. Consistently, the budding yeast SIT4 gene was also a multicopy suppressor for delta ppe1. Phosphatase ppe1 may play a role in cell morphogenesis and mitosis by either regulating or being regulated by these multicopy suppressor gene products. Consistent with this hypothesis, double mutants ppe1-ppa2 and ppe1-pck1 are lethal at the permissive temperature.

scholarly journals Profilin Binding to Poly-l-Proline and Actin Monomers along with Ability to Catalyze Actin Nucleotide Exchange Is Required for Viability of Fission Yeast

2001 ◽  
Vol 12 (4) ◽  
pp. 1161-1175 ◽  
Author(s):  
Jia Lu ◽  
Thomas D. Pollard
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Point Mutations ◽  
Biochemical Properties ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Lower Affinity ◽  
Essential Function

We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-l-proline, but lower affinity for either ligand is incompatible with life; 2) in thecdc3-124 profilin ts background, fission yeast function with profilin having only 2–5% wild-type affinity for actin or poly-l-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-l-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast.

scholarly journals Wobble Inosine tRNA Modification Is Essential to Cell Cycle Progression in G1/S and G2/M Transitions in Fission Yeast

2007 ◽  
Vol 282 (46) ◽  
pp. 33459-33465 ◽  
Author(s):  
Satoshi Tsutsumi ◽  
Reiko Sugiura ◽  
Yan Ma ◽  
Hideki Tokuoka ◽  
Kazuki Ohta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Temperature Sensitive ◽  
Trna Modification ◽  
Gene Encoding ◽  
Cycle Progression ◽  
Catalytically Active ◽  
Mutant Cells

Inosine (I) at position 34 (wobble position) of tRNA is formed by the hydrolytic deamination of a genomically encoded adenosine (A). The enzyme catalyzing this reaction, termed tRNA A:34 deaminase, is the heterodimeric Tad2p/ADAT2·Tad3p/ADAT3 complex in eukaryotes. In budding yeast, deletion of each subunit is lethal, indicating that the wobble inosine tRNA modification is essential for viability; however, most of its physiological roles remain unknown. To identify novel cell cycle mutants in fission yeast, we isolated the tad3-1 mutant that is allelic to the tad3+ gene encoding a homolog of budding yeast Tad3p. Interestingly, the tad3-1 mutant cells principally exhibited cell cycle-specific phenotype, namely temperature-sensitive and irreversible cell cycle arrest both in G1 and G2. Further analyses revealed that in the tad3-1 mutant cells, the S257N mutation that occurred in the catalytically inactive Tad3 subunit affected its association with catalytically active Tad2 subunit, leading to an impairment in the A to I conversion at position 34 of tRNA. In tad3-1 mutant cells, the overexpression of the tad3+ gene completely suppressed the decreased tRNA inosine content. Notably, the overexpression of the tad2+ gene partially suppressed the temperature-sensitive phenotype and the decreased tRNA inosine content, indicating that the tad3-1 mutant phenotype is because of the insufficient I34 formation of tRNA. These results suggest that the wobble inosine tRNA modification is essential for cell cycle progression in the G1/S and G2/M transitions in fission yeast.

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