The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae.

Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.

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The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6789 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6789-6796 ◽

Cited By ~ 60

Author(s):

J Tu ◽

M Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Genetic Analysis ◽

Protein Phosphatase ◽

Glucose Repression ◽

Regulatory Mechanisms ◽

Genetic Studies ◽

Glycogen Accumulation ◽

Regulatory Response

We cloned the GLC7/DIS2S1 gene by complementation of the cid1-226 mutation, which relieves glucose repression in Saccharomyces cerevisiae. GLC7 encodes the catalytic subunit of type 1 protein phosphatase (PP1). Genetic analysis and sequencing showed that cid1-226 is an allele of GLC7, now designated glc7-T152K, which alters threonine 152 to lysine. We also show that the glc7-1 and glc7-T152K alleles cause distinct phenotypes: glc7-1 causes a severe defect in glycogen accumulation but does not relieve glucose repression, whereas glc7-T152K does not prevent glycogen accumulation. These findings are discussed in light of evidence that interaction with different regulatory or targeting subunits directs the participation of PP1 in diverse cellular regulatory mechanisms. Finally, genetic studies suggest that PP1 functions antagonistically to the SNF1 protein kinase in the regulatory response to glucose.

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Saccharomyces cerevisiae Afr1 Protein Is a Protein Phosphatase 1/Glc7-Targeting Subunit That Regulates the Septin Cytoskeleton during Mating

Eukaryotic Cell ◽

10.1128/ec.00024-08 ◽

2008 ◽

Vol 7 (8) ◽

pp. 1246-1255 ◽

Cited By ~ 11

Author(s):

Jennifer P. Bharucha ◽

Jennifer R. Larson ◽

James B. Konopka ◽

Kelly Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Phosphatase ◽

Protein Phosphatase 1 ◽

Auxiliary Subunits ◽

Physiological Pathways ◽

Null Mutants

ABSTRACT Glc7, the type1 serine/threonine phosphatase in the yeast Saccharomyces cerevisiae, is targeted by auxiliary subunits to numerous locations in the cell, where it regulates a range of physiological pathways. We show here that the accumulation of Glc7 at mating projections requires Afr1, a protein required for the formation of normal projections. AFR1-null mutants fail to target Glc7 to projections, and an Afr1 variant specifically defective in binding to Glc7 [Afr1(V546A F548A)] forms aberrant projections. The septin filaments in mating projections of AFR1 mutants initiate normally but then rearrange asymmetrically as the projection develops, suggesting that the Afr1-Glc7 holoenzyme may regulate the maintenance of septin complexes during mating. These results demonstrate a previously unknown role for Afr1 in targeting Glc7 to mating projections and in regulating the septin architecture during mating.

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The Saccharomyces cerevisiae 14-3-3 protein Bmh2 is required for regulation of the phosphorylation status of Fin1, a novel intermediate filament protein

Biochemical Journal ◽

10.1042/bj20020053 ◽

2002 ◽

Vol 365 (1) ◽

pp. 51-56 ◽

Cited By ~ 14

Author(s):

Isabel MAYORDOMO ◽

Pascual SANZ

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Regulation ◽

Intermediate Filament ◽

Protein Phosphatase ◽

Catalytic Subunit ◽

Protein Phosphatase 1 ◽

Intermediate Filament Protein ◽

Filament Protein ◽

Two Hybrid ◽

Hybrid Screening

In order to identify proteins that interact with Bmh2, a yeast member of the 14-3-3 protein family, we performed a two-hybrid screening using LexA-Bmh2 as bait. We identified Fin1, a novel intermediate filament protein, as the protein that showed the highest degree of interaction. We also identified components of the vesicular transport machinery such as Gic2 and Msb3, proteins involved in transcriptional regulation such as Mbf1, Gcr2 and Reg2, and a variety of other different proteins (Ppt1, Lre1, Rps0A and Ylr177w). We studied the interaction between Bmh2 and Fin1 in more detail and found that Bmh2 only interacted with phosphorylated forms of Fin1. In addition, we showed that Glc7, the catalytic subunit of the protein phosphatase 1 complex, was also able to interact with Fin1.

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

The Journal of Cell Biology ◽

10.1083/jcb.149.1.125 ◽

2000 ◽

Vol 149 (1) ◽

pp. 125-140 ◽

Cited By ~ 46

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.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.17.2.620 ◽

1997 ◽

Vol 17 (2) ◽

pp. 620-626 ◽

Cited By ~ 88

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.

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The Saccharomyces cerevisiae SRK1 gene, a suppressor of bcy1 and ins1, may be involved in protein phosphatase function

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.3369-3373.1991 ◽

1991 ◽

Vol 11 (6) ◽

pp. 3369-3373

Author(s):

R B Wilson ◽

A A Brenner ◽

T B White ◽

M J Engler ◽

J P Gaughran ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Cyclic Amp ◽

Protein Phosphatase ◽

Shuttle Vector ◽

Temperature Sensitive ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Cycle Arrest ◽

Dependent Protein

The Saccharomyces cerevisiae SRK1 gene, when expressed on a low-copy shuttle vector, partially suppresses the phenotype associated with elevated levels of cyclic AMP-dependent protein kinase activity and suppresses the temperature-sensitive cell cycle arrest of the ins1 mutant. SRK1 is located on chromosome IV, 3 centimorgans from gcn2. A mutant carrying a deletion mutation in srk1 is viable. SRK1 encodes a 140-kDa protein with homology to the dis3+ protein from Schizosaccharomyces pombe. The ability of SRK1 to alleviate partially the defects caused by high levels of cyclic AMP-dependent protein kinase and the similarity of its encoded protein to dis3+ suggest that SRK1 may have a role in protein phosphatase function.

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Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.5.2226 ◽

1996 ◽

Vol 16 (5) ◽

pp. 2226-2237 ◽

Cited By ~ 300

Author(s):

K W Cunningham ◽

G R Fink

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Phosphatase ◽

Reporter Genes ◽

Immunosuppressive Drug ◽

Ion Pumps ◽

Ion Transporters ◽

Lacz Reporter ◽

Dependent Protein ◽

P Type ◽

Calcineurin Activity

The PMC1 gene in Saccharomyces cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca2+ conditions. Previous work showed that Ca2+ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase sensitive to the immunosuppressive drug FK506. We now report that calcineurin decreases Ca2+ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca2+ exchanger related to vertebrate Na+/Ca2+ exchangers. The contribution of VCX1 in Ca2+ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca2+ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter was slightly diminished and a pmc1::lacZ reporter was induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca2+. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca2+ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca2+ signals. We propose that elevated cytosolic free Ca2+ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions.

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Protein phosphatase type 1 interacts with proteins required for meiosis and other cellular processes in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4199 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4199-4206 ◽

Cited By ~ 67

Author(s):

J Tu ◽

W Song ◽

M Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Hybrid System ◽

Protein Phosphatase ◽

Regulatory Subunit ◽

Specific Substrate ◽

Type I ◽

Cellular Processes ◽

Cell Extracts ◽

Two Hybrid ◽

Protein Phosphatase Type

Protein phosphatase type I (PP1) is involved in diverse cellular processes, and its activity toward specific substrates is thought to be controlled by different regulatory or targeting subunits. To identify regulatory subunits and substrates of the Saccharomyces cerevisiae PP1, encoded by GLC7, we used the two-hybrid system to detect interacting proteins. Among the many proteins identified were Gac1, a known glycogen regulatory subunit, and a protein with homology to Gac1. We also characterized a new gene designated GIP1, for Glc7-interacting protein. We show that a Gip1 fusion protein coimmunoprecipitates with PP1 from cell extracts. Molecular and genetic analyses indicate that GIP1 is expressed specifically during meiosis, affects transcription of late meiotic genes, and is essential for sporulation. Thus, the Gip1 protein is a candidate for a meiosis-specific substrate or regulator of PP1. Finally, we recovered two genes, RED1 and SCD5, with roles in meiosis and the vesicular secretory pathway, respectively. These results provide strong evidence implicating PP1 function in meiosis. In addition, this study indicates that the two-hybrid system offers a promising approach to understanding the multiple roles and interactions of PP1 in cellular regulation.

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