Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis

1991 ◽  
Vol 11 (10) ◽  
pp. 4876-4884
Author(s):  
H Ronne ◽  
M Carlberg ◽  
G Z Hu ◽  
J O Nehlin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Yeast Cells ◽  
Limited Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
The Third ◽  
Phosphatase 2A ◽  
High Level ◽  
Mammalian Protein

We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.

scholarly journals Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis.

1991 ◽  
Vol 11 (10) ◽  
pp. 4876-4884 ◽  
Author(s):  
H Ronne ◽  
M Carlberg ◽  
G Z Hu ◽  
J O Nehlin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Yeast Cells ◽  
Limited Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
The Third ◽  
Phosphatase 2A ◽  
High Level ◽  
Mammalian Protein

We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.

scholarly journals Multiple effects of protein phosphatase 2A on nutrient-induced signalling in the yeast Saccharomyces cerevisiae

2001 ◽  
Vol 40 (4) ◽  
pp. 1020-1026 ◽  
Author(s):  
Ewa Sugajska ◽  
Wojciech Swiatek ◽  
Piotr Zabrocki ◽  
Ilse Geyskens ◽  
Johan M. Thevelein ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Yeast Saccharomyces Cerevisiae ◽  
Phosphatase 2A

scholarly journals Adenovirus Protein E4orf4 Induces Premature APCCdc20 Activation in Saccharomyces cerevisiae by a Protein Phosphatase 2A-Dependent Mechanism

2010 ◽  
Vol 84 (9) ◽  
pp. 4798-4809 ◽  
Author(s):  
Melissa Z. Mui ◽  
Diana E. Roopchand ◽  
Matthew S. Gentry ◽  
Richard L. Hallberg ◽  
Jackie Vogel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Functional Characterization ◽  
Specific Inhibitor ◽  
Human Adenovirus ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
Phosphatase 2A

ABSTRACT Protein phosphatase 2A (PP2A) has been implicated in cell cycle progression and mitosis; however, the complexity of PP2A regulation via multiple B subunits makes its functional characterization a significant challenge. The human adenovirus protein E4orf4 has been found to induce both high Cdk1 activity and the accumulation of cells in G2/M in both mammalian and yeast cells, effects which are largely dependent on the B55/Cdc55 regulatory subunit of PP2A. Thus, E4orf4 represents a unique means by which the function of a specific form of PP2A can be delineated in vivo. In Saccharomyces cerevisiae, only two PP2A regulatory subunits exist, Cdc55 and Rts1. Here, we show that E4orf4-induced toxicity depends on a functional interaction with Cdc55. E4orf4 expression correlates with the inappropriate reduction of Pds1 and Scc1 in S-phase-arrested cells. The unscheduled loss of these proteins suggests the involvement of PP2ACdc55 in the regulation of the Cdc20 form of the anaphase-promoting complex (APC). Contrastingly, activity of the Hct1 form of the APC is not induced by E4orf4, as demonstrated by the observed stability of its substrates. We propose that E4orf4, being a Cdc55-specific inhibitor of PP2A, demonstrates the role of PP2ACdc55 in regulating APCCdc20 activity.

scholarly journals Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (2) ◽  
pp. 620-626 ◽  
Author(s):  
Y Wang ◽  
D J Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Elevated Temperatures ◽  
Protein Phosphatase 2A ◽  
Spindle Checkpoint ◽  
Mitotic Checkpoint ◽  
Regulatory Subunit ◽  
Phosphatase 2A ◽  
A Subunit ◽  
Normal Sensitivity

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.

scholarly journals Cdc55p-Mediated E4orf4 Growth Inhibition in Saccharomyces cerevisiae Is Mediated Only in Part via the Catalytic Subunit of Protein Phosphatase 2A

2008 ◽  
Vol 82 (7) ◽  
pp. 3612-3623 ◽  
Author(s):  
Yikun Li ◽  
Huijun Wei ◽  
Tung-Chin Hsieh ◽  
David C. Pallas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Inhibition ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Point Mutations ◽  
Regulatory Subunit ◽  
Stable Complex ◽  
B Subunit ◽  
C Subunit ◽  
Phosphatase 2A

ABSTRACT The adenovirus early region 4 open reading frame 4 (E4orf4) protein specifically induces p53-independent cell death of transformed but not normal human cells, suggesting that elucidation of its mechanism may provide important new avenues for cancer therapy. Wild-type E4orf4 and mutants that retain cancer cell toxicity also induce growth inhibition in Saccharomyces cerevisiae, which provides a genetically tractable system for studying E4orf4 function. Interaction with the protein phosphatase 2A (PP2A) B regulatory subunit is required for E4orf4's effects, suggesting that E4orf4 may function by regulating B subunit-containing heterotrimeric PP2A holoenzymes (PP2ABAC), which consist of a B subunit complexed with the PP2A structural (A) and catalytic (C) subunits. However, it is not known whether E4orf4-induced growth inhibition requires interaction with the PP2A C subunit or whether E4orf4 might have PP2A B subunit-dependent effects that are independent of PP2ABAC holoenzyme formation. To test these possibilities in S. cerevisiae, we disrupted the stable formation of PP2ABAC heterotrimers and thus E4orf4/C subunit association by PP2A C subunit point mutations or by deletion of the gene for the PP2A methyltransferase, Ppm1p, and assayed for effects on E4orf4-induced growth inhibition. Our results support a model in which E4orf4 mediates growth inhibition and cell killing both through PP2ABAC heterotrimers and through a B regulatory subunit-dependent pathway(s) that is independent of stable complex formation with the PP2A C subunit. They also indicate that Ppm1p has a function other than regulating the assembly of PP2A heterotrimers and suggest that selective PP2A trimer inhibitors and PP6 inhibitors may be useful as adjuvant anticancer therapies.

scholarly journals A Novel Assay for Protein Phosphatase 2A (PP2A) Complexes In Vivo Reveals Differential Effects of Covalent Modifications on Different Saccharomyces cerevisiae PP2A Heterotrimers

2005 ◽  
Vol 4 (6) ◽  
pp. 1029-1040 ◽  
Author(s):  
Matthew S. Gentry ◽  
Yikun Li ◽  
Huijun Wei ◽  
Farhana F. Syed ◽  
Sameer H. Patel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Posttranslational Modifications ◽  
Protein Phosphatase ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Phosphatase 2A ◽  
Covalent Modifications

ABSTRACT Protein phosphatase 2A (PP2A) catalytic subunit can be covalently modified at its carboxy terminus by phosphorylation or carboxymethylation. Determining the effects of these covalent modifications on the relative amounts and functions of different PP2A heterotrimers is essential to understanding how these modifications regulate PP2A-controlled cellular processes. In this study we have validated and used a novel in vivo assay for assessing PP2A heterotrimer formation in Saccharomyces cerevisiae: the measurement of heterotrimer-dependent localization of green fluorescent protein-PP2A subunits. This assay relies on the fact that the correct cellular localization of PP2A requires that it be fully assembled. Thus, reduced localization would occur as the result of the inability to assemble a stable heterotrimer. Using this assay, we determined the effects of PP2A C-subunit phosphorylation mimic mutations and reduction or loss of PP2A methylation on the formation and localization of PP2AB/Cdc55p and PP2AB ′ /Rts1p heterotrimers. Collectively, our findings demonstrate that phosphorylation and methylation of the PP2A catalytic subunit can influence its function both by regulating the total amount of specific PP2A heterotrimers within a cell and by altering the relative proportions of PP2AB/Cdc55p and PP2AB ′ /Rts1p heterotrimers up to 10-fold. Thus, these posttranslational modifications allow flexible, yet highly coordinated, regulation of PP2A-dependent signaling pathways that in turn modulate cell growth and function.

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.

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