scholarly journals Saccharomyces cerevisiae Multidrug Resistance Transporter Qdr2 Is Implicated in Potassium Uptake, Providing a Physiological Advantage to Quinidine-Stressed Cells

2006 ◽  
Vol 6 (2) ◽  
pp. 134-142 ◽  
Author(s):  
Rita C. Vargas ◽  
Raúl García-Salcedo ◽  
Sandra Tenreiro ◽  
Miguel C. Teixeira ◽  
Alexandra R. Fernandes ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Yeast Cell ◽  
Experimental Evidence ◽  
Growth Medium ◽  
Auxotrophic Strain ◽  
General Amino Acid Control ◽  
Stressed Cells ◽  
Multidrug Resistance Transporter

ABSTRACT The QDR2 gene of Saccharomyces cerevisiae encodes a putative plasma membrane drug:H+ antiporter that confers resistance against quinidine, barban, bleomycin, and cisplatin. This work provides experimental evidence of defective K+ (Rb+) uptake in the absence of QDR2. The direct involvement of Qdr2p in K+ uptake is reinforced by the fact that increased K+ (Rb+) uptake due to QDR2 expression is independent of the Trk1p/Trk2p system. QDR2 expression confers a physiological advantage for the yeast cell during the onset of K+ limited growth, due either to a limiting level of K+ in the growth medium or to the presence of quinidine. This drug decreases the K+ uptake rate and K+ accumulation in the yeast cell, especially in the Δqdr2 mutant. Qdr2p also helps to sustain the decrease of intracellular pH in quinidine-stressed cells in growth medium at pH 5.5 by indirectly promoting H+ extrusion affected by the drug. The results are consistent with the hypothesis that Qdr2p may also couple K+ movement with substrate export, presumably with quinidine. Other clues to the biological role of QDR2 in the yeast cell come from two additional lines of experimental evidence. First, QDR2 transcription is activated under nitrogen (NH4 +) limitation or when the auxotrophic strain examined enters stationary phase due to leucine limitation, this regulation being dependent on general amino acid control by Gcn4p. Second, the amino acid pool is higher in Δqdr2 cells than in wild-type cells, indicating that QDR2 expression is, directly or indirectly, involved in amino acid homeostasis.

scholarly journals Role of Integrase in Reverse Transcription of the Saccharomyces cerevisiae Retrotransposon Ty1

2005 ◽  
Vol 4 (6) ◽  
pp. 1057-1065 ◽  
Author(s):  
M. Wilhelm ◽  
F.-X. Wilhelm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Specific Activity ◽  
Rnase H ◽  
Amino Acid Residues ◽  
Acidic Amino Acid ◽  
Acidic Domain ◽  
Basic Region

ABSTRACT Reverse transcriptase (RT) with its associated RNase H (RH) domain and integrase (IN) are key enzymes encoded by retroviruses and retrotransposons. Several studies have implied a functional role of the interaction between IN and RT during the replication of retroviral and retrotransposon genomes. In this study, IN deletion mutants were used to investigate the role of IN on the RT activity of the yeast Saccharomyces cerevisiae retrotransposon Ty1. We have identified two domains of Ty1 integrase which have effects on RT activity in vivo. The deletion of a domain spanning amino acid residues 233 to 520 of IN increases the exogenous specific activity of RT up to 20-fold, whereas the removal of a region rich in acidic amino acid residues between residues 521 and 607 decreases its activity. The last result complements our observation that an active recombinant RT protein can be obtained if a small acidic tail mimicking the acidic domain of IN is fused to the RT-RH domain. We suggest that interaction between these acidic amino acid residues of IN and a basic region of RT could be critical for the correct folding of RT and for the formation of an active conformation of the enzyme.

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

1986 ◽  
Vol 32 (12) ◽  
pp. 969-972 ◽  
Author(s):  
Albert J. Wilson ◽  
J. K. Bhattacharjee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Kinase ◽  
Growth Medium ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Carbon Sources ◽  
Absolute Requirement ◽  
Fructose 1,6 Bisphosphatase ◽  
Futile Cycling ◽  
Respiratory Deficient

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present sumultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.

scholarly journals Role of HSP90 in Salt Stress Tolerance via Stabilization and Regulation of Calcineurin

2000 ◽  
Vol 20 (24) ◽  
pp. 9262-9270 ◽  
Author(s):  
Jun Imai ◽  
Ichiro Yahara
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Salt Stress ◽  
Stress Tolerance ◽  
Normal Level ◽  
Immune Complexes ◽  
Salt Stress Tolerance ◽  
Stressed Cells ◽  
Calcineurin Activity

ABSTRACT The role of HSP90 in stress tolerance was investigated inSaccharomyces cerevisiae. Cells showing 20-fold overexpression of Hsc82, an HSP90 homologue in yeast, were hypersensitive to high-NaCl or H-LiCl stresses. Hsc82-overexpressing cells appeared similar to calcineurin-defective cells in salt sensitivity and showed reduced levels of calcineurin-dependent gene expression. Co-overexpression of Cna2, the catalytic subunit of calcineurin, suppressed the hypersensitivity. Cna2 and Hsc82 coimmunoprecipitated from control cells grown under normal conditions but not from stressed cells. In contrast, coimmunoprecipitation was detected with Hsc82-overexpressing cells even after exposure to stresses. Cna2 immune complexes from stressed control cells showed a significant level of calcineurin activity, whereas those from stressed Hsc82-overexpressing cells did not. Treatment of extracts from Hsc82-overexpressing cells with Ca2+-calmodulin increased the calcineurin activity associated with Cna2 immune complexes. Geldanamycin, an inhibitor of HSP90 abolished the coimmunoprecipitation but did not activate calcineurin. When the expression level of Hsc82 decreased to below 30% of the normal level, cells also became hypersensitive to salt stress. In these cells, the amount of Cna2 was reduced, likely as a result of degradation. The present results showed that Hsc82 binds to and stabilizes Cna2 and that dissociation of Cna2 from Hsc82 is necessary for its activation.

The yeast ILV2 gene is under general amino acid control

Genome ◽  
1988 ◽  
Vol 30 (6) ◽  
pp. 984-986 ◽  
Author(s):  
W. Xiao ◽  
G. H. Rank
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Key Words ◽  
Acetolactate Synthase ◽  
Amino Acid Starvation ◽  
Sulfometuron Methyl ◽  
General Amino Acid Control ◽  
Acid Control ◽  
High Level ◽  
Level Resistance

The yeast ILV2 gene encodes acetolactate synthase, the first enzyme in the biosynthesis of isoleucine and valine. Its multiple regulation has precluded the clear demonstration of whether ILV2 is under general amino acid control. Nonderepressible gcn4 strains were used as recipients for transformation with a YCp plasmid carrying GCN4. Parental gcn4 cells and their isogenic GCN4 transformants were evaluated for ALS derepression following induced amino acid starvation. GCN4 cells showed 1.5-to 1.7-fold derepression but no derepression was observed in isogenic control gcn4 strains. A similar depression of ILV2 mRNA was also observed. Genetic evidence for general amino acid control was the gcn4 suppression of high level resistance to sulfometuron methyl by the SMR1-410 allele of ILV2.Key words: Saccharomyces cerevisiae, ILV2 gene, general amino acid control, multiple regulators.

scholarly journals General amino acid control and specific arginine repression in Saccharomyces cerevisiae: physical study of the bifunctional regulatory region of the ARG3 gene.

1985 ◽  
Vol 5 (11) ◽  
pp. 3139-3148 ◽  
Author(s):  
M Crabeel ◽  
R Huygen ◽  
K Verschueren ◽  
F Messenguy ◽  
K Tinel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Regulatory Region ◽  
Positive Control ◽  
Posttranscriptional Control ◽  
General Amino Acid Control ◽  
Acid Control ◽  
Physical Study ◽  
Specific Regulation ◽  
General Regulation

To characterize further the regulatory mechanism modulating the expression of the Saccharomyces cerevisiae ARG3 gene, i.e., the specific repression by arginine and the general amino acid control, we analyzed by deletion the region upstream of that gene, determined the nucleotide sequence of operator-constitutive-like mutations affecting the specific regulation, and examined the behavior of an ARG3-galK fusion engineered at the initiating codon of ARG3. Similarly to what was observed in previous studies on the HIS3 and HIS4 genes, our data show that the general regulation acts as a positive control and that a sequence containing the nucleotide TGACTC, between positions -364 and -282 upstream of the transcription start, functions as a regulatory target site. This sequence contains the most proximal of the two TGACTC boxes identified in front of ARG3. While the general control appears to modulate transcription efficiency, the specific repression by arginine displays a posttranscriptional component (F. Messenguy and E. Dubois, Mol. Gen. Genet. 189:148-156, 1983). Our deletion and gene fusion analyses confirm that the specific and general controls operate independently of each other and assign the site responsible for arginine-specific repression to between positions -170 and +22. In keeping with this assignment, the two operator-constitutive-like mutations were localized at positions -80 and -46, respectively, and thus in a region which is not transcribed. We discuss a hypothesis accounting for the involvement of untranscribed DNA in a posttranscriptional control.

scholarly journals Biological role of the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

1981 ◽  
Vol 1 (7) ◽  
pp. 584-593 ◽  
Author(s):  
P Niederberger ◽  
G Miozzari ◽  
R Hütter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Biosynthesis ◽  
Wild Type ◽  
General Control ◽  
Acid Biosynthesis ◽  
Mutant Strains ◽  
In The Wild ◽  
Amino Acid Imbalance

The biological role of the "general control of amino acid biosynthesis" has been investigated by analyzing growth and enzyme levels in wild-type, bradytrophic, and nonderepressing mutant strains of Saccharomyces cerevisiae. Amino acid limitation was achieved by using either bradytrophic mutations or external amino acid imbalance. In the wild-type strain noncoordinate derepression of enzymes subject to the general control has been found. Derepressing factors were in the order of 2 to 4 in bradytrophic mutant strains grown under limiting conditions and only in the order of 1.5 to 2 under the influence of external amino acid imbalance. Nonderepressing mutations led to slower growth rates under conditions of amino acid limitation, and no derepression of enzymes under the general control was observed. The amino acid pools were found to be very similar in the wild type and in nonderepressing mutant strains under all conditions tested. Our results indicate that the general control affects all branched amino acid biosynthetic pathways, namely, those of the aromatic amino acids and the aspartate family, the pathways for the basic amino acids lysine, histidine, and arginine, and also the pathways of serine and valine biosyntheses.

scholarly journals Hypomodified tRNA in evolutionarily distant yeasts can trigger rapid tRNA decay to activate the general amino acid control response, but with different consequences

2020 ◽  
Author(s):  
Thareendra De Zoysa ◽  
Eric M. Phizicky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Amino Acid ◽  
Temperature Sensitivity ◽  
Temperature Sensitive ◽  
Temperature Resistance ◽  
General Amino Acid Control ◽  
Growth Defects ◽  
Acid Control ◽  
Body Modifications

AbstractAll tRNAs are extensively modified, and modification deficiency often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of any of several tRNA body modifications results in rapid tRNA decay (RTD) of certain mature tRNAs by the 5’-3’ exonucleases Rat1 and Xrn1. As tRNA quality control decay mechanisms are not extensively studied in other eukaryotes, we studied trm8Δ mutants in the evolutionarily distant fission yeast Schizosaccharomyces pombe, which lack 7-methylguanosine at G46 of tRNAs. We report here that S. pombe trm8Δ mutants are temperature sensitive primarily due to decay of tRNATyr(GUA) and that spontaneous mutations in the RAT1 ortholog dhp1+ restored temperature resistance and prevented tRNA decay, demonstrating conservation of the RTD pathway. We also report for the first time evidence linking the RTD and the general amino acid control (GAAC) pathways, which we show in both S. pombe and S. cerevisiae. In S. pombe trm8Δ mutants, spontaneous GAAC mutations restored temperature resistance and tRNA levels, and the temperature sensitivity of trm8Δ mutants was precisely linked to GAAC activation due to tRNATyr(GUA) decay. Similarly, in the well-studied S. cerevisiae trm8Δ trm4Δ RTD mutant, temperature sensitivity was closely linked to GAAC activation due to tRNAVal(AAC) decay; however, in S. cerevisiae, GAAC mutations increased tRNA decay and enhanced temperature sensitivity. Thus, these results demonstrate a conserved GAAC activation coincident with RTD in S. pombe and S. cerevisiae, but an opposite impact of the GAAC response in the two organisms. We speculate that the RTD pathway and its regulation of the GAAC pathway is widely conserved in eukaryotes, extending to other mutants affecting tRNA body modifications.Author SummarytRNA modifications are highly conserved and their lack frequently results in growth defects in the yeast Saccharomyces cerevisiae and neuorological disorders in humans. S. cerevsiaie has two tRNA quality control decay pathways that sense tRNAs lacking modifications in the main tRNA body. One of these, the rapid tRNA decay (RTD) pathway, targets mature tRNAs for 5’-3’ exonucleolytic decay by Rat1 and Xrn1. It is unknown if RTD is conserved in eukaryotes, and if it might explain phenotypes associated with body modification defects. Here we focus on trm8Δ mutants, lacking m7G46, in the evolutionarily distant yeast Schizosaccharomyces pombe. Loss of m7G causes temperature sensitivity and RTD in S. cerevisiae, microcephalic primordial dwarfism in humans, and defective stem cell renewal in mice. We show that S. pombe trm8Δ mutants are temperature sensitive due to tY(GUA) decay by Rat1, implying conservation of RTD among divergent eukaryotes. We also show that the onset of RTD triggers activation of the general amino acid control (GAAC) pathway in both S. pombe and S. cerevisiae, resulting in exacerbated decay in S. pombe and reduced decay in S. cerevisiae. We speculate that RTD and its regulation of the GAAC pathway will be widely conserved in eukaryotes including humans.

scholarly journals Arginine restriction induced by delta-N-(phosphonacetyl)-L-ornithine signals increased expression of HIS3, TRP5, CPA1, and CPA2 in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (11) ◽  
pp. 4882-4888 ◽  
Author(s):  
D M Kinney ◽  
C J Lusty
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translational Control ◽  
Mrna Translation ◽  
Single Amino Acid ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
General Amino Acid Control ◽  
Acid Control ◽  
Beta Galactosidase

delta-N-(Phosphonacetyl)-L-ornithine (PALO), a transition state analog inhibitor of ornithine transcarbamylase, induced arginine limitation in vivo in Saccharomyces cerevisiae. Arginine restriction caused increased expression of HIS3 and TRP5, measured by the beta-galactosidase activity in strains carrying chromosomally integrated fusions of the promoter regions of each gene with the lacZ gene of Escherichia coli. The increase in beta-galactosidase activity induced by PALO was reversed by the addition of arginine and was dependent on GCN4 protein. These results indicate that PALO, like 3-amino-1,2,4-triazole DL-5-methyltryptophan, can be used to study the effect of limitation of a single amino acid, arginine, on the expression of genes under the general amino acid control regulatory system. Arginine deprivation imposed by PALO also caused increased expression of CPA1 and CPA2, coding respectively for the small and large subunits of arginine-specific carbamyl-phosphate synthetase. The observed increase was GCN4 dependent and was genetically separable from arginine-specific repression of CPA1 mRNA translation. The 5'-flanking regions of CPA1 (reported previously) and CPA2 determined in this study each contained at least two copies of the sequence TGACTC, shown to bind GCN4 protein. The beta-galactosidase activities expressed from CPA1- and CPA2-lacZ fusions integrated into the nuclear DNA of gcn4 mutant strains were five to six times less than in the wild type, when both strains were grown under depressed conditions. The gcn4 mutation reduced basal expression of both CPA1 and CPA2. The addition of arginine to strains containing the CPA1-lacZ fusion further reduced beta-galactosidase activity of the gcn4 mutant, indicating independent regulation of the CPA1 gene by the general amino acid control and by arginine-specific repression. In strains overproducing GCN4 protein, the translational control completely overrode transcriptional activation of CPA1 by general amino acid control.

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