Heat Stress Regulates the Expression of TPK1 Gene at Transcriptional and Post-Transcriptional Levels in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, cAMP regulates a number of different cellular processes, such as cell growth, metabolism, stress resistance and gene transcription. The intracellular target for this second messenger in yeast cells is the cAMP-dependent protein kinase (PKA). The way in which a broad specificity protein kinase mediates one right physiological response after cAMP increase indicates that specificity is highly regulated in the cAMP / PKA system. Here we address the mechanism through which cAMP-PKA signalling mediates its response to heat shock thermotolerance in Saccharomyces cerevisiae. Yeast PKA is a tetrameric holoenzyme composed of a regulatory (Bcy1) subunit dimer and two catalytic subunits (Tpk1, Tpk2 and Tpk3). PKA subunits are differentially expressed under certain stress conditions. In the present study we show that, although the mRNA levels of TPK1 are upregulated upon heat shock at 37℃, no change is detected in Tpk1 protein levels. The half-life of TPK1 mRNA increases and this mRNA condensates in cytoplasmic foci upon thermal stress. The resistance of TPK1 mRNA foci to cycloheximide-induced disassembly, together with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 mRNA foci and TPK1 expression were also evaluated during thermotolerance. The crosstalk of cAMP-PKA pathway and cell wall integrity (CWI) signalling was also studied. Wsc3 sensor and other components of the CWI pathway are necessary for the upregulation of TPK1 mRNA upon heat shock conditions. The assembly in cytoplasmic foci upon thermal stress shows to be dependent of Wsc3. Finally, evidence of an increase in the abundance of Tpk1 in the PKA holoenzyme in response to heat shock is presented. The results indicate the existence of a mechanism that exclusively regulates Tpk1 subunit expression, which contributes to cAMP-PKA specificity and also suggest that a recurrent stress enhanced the fitness for the coming favorable conditions.

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Btn2 is involved in the clearance of denatured proteins caused by severe ethanol stress in Saccharomyces cerevisiae

FEMS Yeast Research ◽

10.1093/femsyr/foz079 ◽

2019 ◽

Cited By ~ 3

Author(s):

Sae Kato ◽

Masashi Yoshida ◽

Shingo Izawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Protein Quality ◽

Protein Denaturation ◽

Recovery Process ◽

Ubiquitin Proteasome System ◽

Yeast Cells ◽

Ethanol Stress ◽

Protein Levels ◽

Denatured Proteins

Abstract Saccharomyces cerevisiae shows similar responses to heat shock and ethanol stress. Cells treated with severe ethanol stress activate the transcription of HSP genes and cause the aggregation of Hsp104-GFP, implying that severe ethanol stress as well as heat shock causes the accumulation of denatured proteins in yeast cells. However, there is currently no concrete evidence to show that severe ethanol stress causes protein denaturation in living yeast cells. In the present study, we investigated whether severe ethanol stress causes protein denaturation, and confirmed that a treatment with 10% (v/v) ethanol stress resulted in the accumulation of insoluble proteins and ubiquitinated proteins in yeast cells. We also found that increased denatured protein levels were efficiently reduced by the ubiquitin-proteasome system after the elimination of ethanol. Since our previous findings demonstrated that the expression of Btn2 was induced by severe ethanol stress, we herein examined the importance of Btn2 in protein quality control in cells treated with severe ethanol stress. btn2∆ cells showed a significant delay in the clearance of denatured proteins during the recovery process. These results provide further insights into the effects of severe ethanol on yeast proteostasis and the contribution of Btn2 to the efficient clearance of denatured proteins.

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The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A.

Molecular and Cellular Biology ◽

10.1128/mcb.15.11.6232 ◽

1995 ◽

Vol 15 (11) ◽

pp. 6232-6245 ◽

Cited By ~ 136

Author(s):

J C Varela ◽

U M Praekelt ◽

P A Meacock ◽

R J Planta ◽

W H Mager

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Protein Kinase ◽

Stationary Phase ◽

Protein Kinase A ◽

Yeast Cells ◽

Wild Type ◽

High Osmolarity ◽

High Osmolarity Glycerol ◽

Kinase A

The HSP12 gene encodes one of the two major small heat shock proteins of Saccharomyces cerevisiae. Hsp12 accumulates massively in yeast cells exposed to heat shock, osmostress, oxidative stress, and high concentrations of alcohol as well as in early-stationary-phase cells. We have cloned an extended 5'-flanking region of the HSP12 gene in order to identify cis-acting elements involved in regulation of this highly expressed stress gene. A detailed analysis of the HSP12 promoter region revealed that five repeats of the stress-responsive CCCCT motif (stress-responsive element [STRE]) are essential to confer wild-type induced levels on a reporter gene upon osmostress, heat shock, and entry into stationary phase. Disruption of the HOG1 and PBS2 genes leads to a dramatic decrease of the HSP12 inducibility in osmostressed cells, whereas overproduction of Hog1 produces a fivefold increase in wild-type induced levels upon a shift to a high salt concentration. On the other hand, mutations resulting in high protein kinase A (PKA) activity reduce or abolish the accumulation of the HSP12 mRNA in stressed cells. Conversely, mutants containing defective PKA catalytic subunits exhibit high basal levels of HSP12 mRNA. Taken together, these results suggest that HSP12 is a target of the high-osmolarity glycerol (HOG) response pathway under negative control of the Ras-PKA pathway. Furthermore, they confirm earlier observations that STRE-like sequences are responsive to a broad range of stresses and that the HOG and Ras-PKA pathways have antagonistic effects upon CCCCT-driven transcription.

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Repetitive Dictyostelium heat-shock promotor functions in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.4.4.591-598.1984 ◽

1984 ◽

Vol 4 (4) ◽

pp. 591-598

Author(s):

J Cappello ◽

C Zuker ◽

H F Lodish

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Nucleotide Sequence ◽

Dna Sequence ◽

Base Pair ◽

Shock Treatment ◽

Yeast Cells ◽

Genomic Segment ◽

Heat Shock Treatment ◽

Shock Response

The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.

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Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2914-2924.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2914-2924

Author(s):

A Hoekema ◽

R A Kastelein ◽

M Vasser ◽

H A de Boer

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Steady State ◽

Codon Usage ◽

Experimental Approach ◽

Mrna Translation ◽

Yeast Cells ◽

Expression Level ◽

Start Codon ◽

Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

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MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.3076-3083.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 3076-3083

Author(s):

K Irie ◽

M Takase ◽

K S Lee ◽

D E Levin ◽

H Araki ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase C ◽

Protein Kinase ◽

Map Kinase ◽

Protein Kinases ◽

Cell Lysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Kinase C ◽

Mitogen Activated Protein

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.

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Screening for Modulators of Spermine Tolerance Identifies Sky1, the SR Protein Kinase of Saccharomyces cerevisiae, as a Regulator of Polyamine Transport and Ion Homeostasis

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.175-184.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 175-184 ◽

Cited By ~ 30

Author(s):

Omri Erez ◽

Chaim Kahana

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Osmotic Shock ◽

Ion Homeostasis ◽

Inorganic Ions ◽

Yeast Cells ◽

Wild Type ◽

Sr Protein ◽

Polyamine Transport ◽

Increased Sensitivity

ABSTRACT Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown. Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants ofSaccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport. Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine. Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine. SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine. In addition to spermine tolerance, sky1Δ cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock. The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport. We demonstrate that these two kinases act in two different signaling pathways. While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity ofPPZ1 but not of ENA1.

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Repetitive Dictyostelium heat-shock promotor functions in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.4.4.591 ◽

1984 ◽

Vol 4 (4) ◽

pp. 591-598 ◽

Cited By ~ 10

Author(s):

J Cappello ◽

C Zuker ◽

H F Lodish

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Nucleotide Sequence ◽

Dna Sequence ◽

Base Pair ◽

Shock Treatment ◽

Yeast Cells ◽

Genomic Segment ◽

Heat Shock Treatment ◽

Shock Response

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Regulation of STA1 gene expression by MAT during the life cycle of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3992-3998.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3992-3998

Author(s):

A M Dranginis

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Yeast Cells ◽

Specific Gene ◽

Activity Levels ◽

Sporulation Medium ◽

Mrna Levels ◽

Yeast Saccharomyces Cerevisiae ◽

Type Locus ◽

Diploid Cells

STA1 encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var. diastaticus. Glucoamylase secretion is controlled by the mating type locus MAT; a and alpha haploid yeast cells secrete high levels of the enzyme, but a/alpha diploid cells produce undetectable amounts. It has been suggested that STA1 is regulated by MATa2 (I. Yamashita, Y. Takano, and S. Fukui, J. Bacteriol. 164:769-773, 1985), which is a MAT transcript of previously unknown function. In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA1 regulation but that deletion of MATa1 sequences completely abolished mating-type control. In all cases, glucoamylase activity levels reflected STA1 mRNA levels. It appears that STA1 is a haploid-specific gene that is regulated by MATa1 and a product of the MAT alpha locus and that this regulation occurs at the level of RNA accumulation. STA1 expression was also shown to be glucose repressible. STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulation-specific glucoamylase of S. cerevisiae. A diploid strain with a MATa1 deletion showed normal induction of STA1 in sporulation medium, but SGA expression was abolished. Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation. STA1 induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation. The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally.

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The yeast EUG1 gene encodes an endoplasmic reticulum protein that is functionally related to protein disulfide isomerase

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4601-4611.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4601-4611

Author(s):

C Tachibana ◽

T H Stevens

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Protein Disulfide Isomerase ◽

Yeast Cells ◽

Disulfide Isomerase ◽

Protein Levels ◽

Growth Defects ◽

Endoplasmic Reticulum Protein ◽

Related Proteins ◽

Protein Disulfide

The product of the EUG1 gene of Saccharomyces cerevisiae is a soluble endoplasmic reticulum protein with homology to both the mammalian protein disulfide isomerase (PDI) and the yeast PDI homolog encoded by the essential PDI1 gene. Deletion or overexpression of EUG1 causes no growth defects under a variety of conditions. EUG1 mRNA and protein levels are dramatically increased in response to the accumulation of native or unglycosylated proteins in the endoplasmic reticulum. Overexpression of the EUG1 gene allows yeast cells to grow in the absence of the PDI1 gene product. Depletion of the PDI1 protein in Saccharomyces cerevisiae causes a soluble vacuolar glycoprotein to accumulate in its endoplasmic reticulum form, and this phenotype is only partially relieved by the overexpression of EUG1. Taken together, our results indicate that PDI1 and EUG1 encode functionally related proteins that are likely to be involved in interacting with nascent polypeptides in the yeast endoplasmic reticulum.

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Starvation promotes Dictyostelium development by relieving PufA inhibition of PKA translation through the YakA kinase pathway

Development ◽

10.1242/dev.126.14.3263 ◽

1999 ◽

Vol 126 (14) ◽

pp. 3263-3274 ◽

Cited By ~ 3

Author(s):

G.M. Souza ◽

A.M. da Silva ◽

A. Kuspa

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Mrna Levels ◽

Wild Type ◽

Mobility Shift ◽

Developmental Defects ◽

C Protein ◽

Protein Levels ◽

Cell Extracts ◽

Puf Protein

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.

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