Cell-Cycle Perturbations Suppress the Slow-Growth Defect of spt10Δ Mutants in Saccharomyces cerevisiae

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.

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Depolarization of the actin cytoskeleton is a specific phenotype in Saccharomyces cerevisiae

Journal of Cell Science ◽

10.1242/jcs.111.17.2689 ◽

1998 ◽

Vol 111 (17) ◽

pp. 2689-2696 ◽

Cited By ~ 2

Author(s):

T.S. Karpova ◽

S.L. Moltz ◽

L.E. Riles ◽

U. Guldener ◽

J.H. Hegemann ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Environmental Factors ◽

Actin Cytoskeleton ◽

Slow Growth ◽

Specific Response ◽

Number Of Genes ◽

Chromosome I ◽

Harsh Conditions

The yeast actin cytoskeleton is polarized during most of the cell cycle. Certain environmental factors and mutations are associated with depolarization of the actin cytoskeleton. Is depolarization of the actin cytoskeleton a specific response, or is it a nonspecific reaction to harsh conditions or poor metabolism? If depolarization is a nonspecific response, then any mutation that slows growth should induce depolarization. In addition, the number of genes with the depolarization phenotype should constitute a relatively large part of the genome. To address this question, we determined the effect of slow growth on the actin cytoskeleton, and we determined the frequency of mutations that affect the actin cytoskeleton. Eight mutants with slow growth showed no defect in actin polarization, indicating that slow growth alone is not sufficient to cause depolarization. Among 273 viable haploids disrupted for ORFs of chromosome I and VIII and 950 viable haploids with random genome disruptions, none had depolarization of the cytoskeleton. We conclude that depolarization of the actin cytoskeleton is a specific phenotype.

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The REG2 gene of Saccharomyces cerevisiae encodes a type 1 protein phosphatase-binding protein that functions with Reg1p and the Snf1 protein kinase to regulate growth.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2922 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2922-2931 ◽

Cited By ~ 47

Author(s):

D L Frederick ◽

K Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Protein Phosphatase ◽

Slow Growth ◽

Glucose Repression ◽

Growth Defect ◽

Loss Of Function ◽

Cell Extracts

The GLC7 gene of Saccharomyces cerevisiae encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is essential for cell growth. We have isolated a previously uncharacterized gene, REG2, on the basis of its ability to interact with Glc7p in the two-hybrid system. Reg2p interacts with Glc7p in vivo, and epitope-tagged derivatives of Reg2p and Glc7p coimmunoprecipitate from cell extracts. The predicted protein product of the REG2 gene is similar to Reg1p, a protein believed to direct PP1 activity in the glucose repression pathway. Mutants with a deletion of reg1 display a mild slow-growth defect, while reg2 mutants exhibit a wild-type phenotype. However, mutants with deletions of both reg1 and reg2 exhibit a severe growth defect. Overexpression of REG2 complements the slow-growth defect of a reg1 mutant but does not complement defects in glycogen accumulation or glucose repression, two traits also associated with a reg1 deletion. These results indicate that REG1 has a unique role in the glucose repression pathway but acts together with REG2 to regulate some as yet uncharacterized function important for growth. The growth defect of a reg1 reg2 double mutant is alleviated by a loss-of-function mutation in the SNF1-encoded protein kinase. The snf1 mutation also suppresses the glucose repression defects of reg1. Together, our data are consistent with a model in which Reg1p and Reg2p control the activity of PP1 toward substrates that are phosphorylated by the Snf1p kinase.

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The Yeast Ser/Thr Phosphatases Sit4 and Ppz1 Play Opposite Roles in Regulation of the Cell Cycle

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2408 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2408-2415 ◽

Cited By ~ 56

Author(s):

Josep Clotet ◽

Eloi Garí ◽

Martí Aldea ◽

Joaquín Ariño

Keyword(s):

Cell Cycle ◽

Protein Phosphatase ◽

Slow Growth ◽

Negative Regulator ◽

Yeast Cells ◽

Growth Defect ◽

Gene Encoding ◽

Synthetic Lethal ◽

Bud Emergence ◽

Normal Transition

ABSTRACT Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from α-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 onsit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression ofPPZ1 is intensified in strains with low G1cyclin levels (such as bck2Δ or cln3Δ mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition.

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TOR2 Is Part of Two Related Signaling Pathways Coordinating Cell Growth in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/148.1.99 ◽

1998 ◽

Vol 148 (1) ◽

pp. 99-112 ◽

Cited By ~ 3

Author(s):

Stephen B Helliwell ◽

Isabelle Howald ◽

Nik Barbet ◽

Michael N Hall

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Protein Synthesis ◽

Cell Growth ◽

Actin Cytoskeleton ◽

Signaling Pathways ◽

Growth Defect ◽

G1 Arrest ◽

Class A ◽

Class B

Abstract The Saccharomyces cerevisiae genes TOR1 and TOR2 encode phosphatidylinositol kinase homologs. TOR2 has two essential functions. One function overlaps with TOR1 and mediates protein synthesis and cell cycle progression. The second essential function of TOR2 is unique to TOR2 and mediates the cell-cycle-dependent organization of the actin cytoskeleton. We have isolated temperature-sensitive mutants that are defective for either one or both of the two TOR2 functions. The three classes of mutants were as follows. Class A mutants, lacking only the TOR2-unique function, are defective in actin cytoskeleton organization and arrest within two to three generations as small-budded cells in the G2/M phase of the cell cycle. Class B mutants, lacking only the TOR-shared function, and class C mutants, lacking both functions, exhibit a rapid loss of protein synthesis and a G1 arrest within one generation. To define further the two functions of TOR2, we isolated multicopy suppressors that rescue the class A or B mutants. Overexpression of MSS4, PKC1, PLC1, RHO2, ROM2, or SUR1 suppressed the growth defect of a class A mutant. Surprisingly, overexpression of PLC1 and MSS4 also suppressed the growth defect of a class B mutant. These genes encode proteins that are involved in phosphoinositide metabolism and signaling. Thus, the two functions (readouts) of TOR2 appear to involve two related signaling pathways controlling cell growth.

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HTL1 Encodes a Novel Factor That Interacts with the RSC Chromatin Remodeling Complex in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.22.23.8165-8174.2002 ◽

2002 ◽

Vol 22 (23) ◽

pp. 8165-8174 ◽

Cited By ~ 22

Author(s):

Martin J. Romeo ◽

Melinda L. Angus-Hill ◽

Andrew K. Sobering ◽

Yoshiaki Kamada ◽

Bradley R. Cairns ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Protein Kinase C ◽

Protein Kinase ◽

Chromatin Remodeling ◽

Growth Defect ◽

Growth Defects ◽

Kinase C ◽

Chromatin Remodeling Complex ◽

Rsc Complex

ABSTRACT RSC is an essential chromatin remodeling complex in Saccharomyces cerevisiae that performs central roles in transcriptional regulation and cell cycle progression. Here we identify Htl1 as a novel factor that associates with the RSC complex both physically and functionally. We isolated HTL1 through a genetic screen for mutants that displayed additive growth defects with a conditional mutation in the protein kinase C gene (PKC1), which has been suggested through genetic connections to interact functionally with RSC. Several lines of evidence connect HTL1 to RSC function. First, an htl1Δ mutant displayed temperature-sensitive growth and a G2/M cell cycle arrest at restrictive temperatures, a phenotype similar to that of strains with conditional mutations in essential RSC components. Second, we isolated RSC3, which encodes a component of the RSC complex, as a dosage suppressor of the htl1Δ growth arrest. Third, an htl1Δ mutant displayed additive growth defects with conditional rsc3 alleles. Fourth, overexpression of HTL1 suppressed the growth defect of a strain with a conditional mutation in another RSC component, RSC8. Finally, we demonstrate that Htl1 is a nuclear protein that can associate in vivo with a fraction of the RSC complex. We propose that an RSC-Htl1 complex acts coordinately with protein kinase C to regulate the G2/M transition.

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The Glc7 type 1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M.

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3158 ◽

1994 ◽

Vol 14 (5) ◽

pp. 3158-3165 ◽

Cited By ~ 62

Author(s):

N Hisamoto ◽

K Sugimoto ◽

K Matsumoto

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Protein Phosphatase ◽

Cold Sensitivity ◽

Growth Defect ◽

Protein Kinase Activity ◽

Restrictive Temperature ◽

Single Mutation ◽

Permissive Temperature

We isolated a mutant carrying a conditional mutation in the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by selection of suppressors that restored the growth defect of cdc24 mutants at high temperature and simultaneously conferred cold-sensitive growth. This cold sensitivity for growth is caused by a single mutation (glc7Y-170) at position 170 of the Glc7 protein, resulting in replacement of cysteine with tyrosine. Genetic analysis suggested that the glc7Y-170 allele is associated with a recessive negative phenotype, reducing the activity of Glc7 in the cell. The glc7Y-170 mutant missegregated chromosome III at the permissive temperature, arrested growth as large-budded cells at the restrictive temperature, exhibited a significant increase in the number of nuclei at or in the neck, and had a short spindle. Furthermore, the glc7Y-170 mutant exhibited a high level of CDC28-dependent protein kinase activity when incubated at the restrictive temperature. These findings suggest that the glc7Y-170 mutation is defective in the G2/M phase of the cell cycle. Thus, type 1 protein phosphatase in Saccharomyces cerevisiae is essential for the G2/M transition.

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The Short Life Span of Saccharomyces cerevisiae sgs1 and srs2 Mutants Is a Composite of Normal Aging Processes and Mitotic Arrest Due to Defective Recombination

Genetics ◽

10.1093/genetics/157.4.1531 ◽

2001 ◽

Vol 157 (4) ◽

pp. 1531-1542 ◽

Cited By ~ 4

Author(s):

Mitch McVey ◽

Matt Kaeberlein ◽

Heidi A Tissenbaum ◽

Leonard Guarente

Keyword(s):

Cell Cycle ◽

Life Span ◽

Mitotic Cell ◽

Dna Helicase ◽

Premature Aging ◽

Growth Defect ◽

Short Life Span ◽

Single Strand Annealing ◽

Late Generation ◽

Strand Annealing

Abstract Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability. Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging. Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background. In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants. Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena. A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells. This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint. Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles. Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest. This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination. Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.

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Underproduction of the Largest Subunit of RNA Polymerase II Causes Temperature Sensitivity, Slow Growth, and Inositol Auxotrophy in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.3.737 ◽

1996 ◽

Vol 142 (3) ◽

pp. 737-747 ◽

Cited By ~ 7

Author(s):

Jacques Archambault ◽

David B Jansma ◽

James D Friesen

Keyword(s):

Saccharomyces Cerevisiae ◽

Steady State ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Reporter Gene ◽

Growth Medium ◽

Temperature Sensitivity ◽

Slow Growth ◽

Yeast Saccharomyces Cerevisiae ◽

Genes Encoding

Abstract In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery.

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

Genetics ◽

10.1093/genetics/140.1.67 ◽

1995 ◽

Vol 140 (1) ◽

pp. 67-77 ◽

Cited By ~ 1

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.

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