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.

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.

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 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.

Identification of GCD14 and GCD15, Novel Genes Required for Translational Repression of GCN4 mRNA in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 1007-1020 ◽  
Author(s):  
Rafael Cuesta ◽  
Alan G Hinnebusch ◽  
Mercedes Tamame
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translation Initiation ◽  
Translational Control ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mrna Levels ◽  
New Genes ◽  
General Translation

Abstract In Saccharomyces cerevisiae, expression of the transcriptional activator GCN4 increases at the translational level in response to starvation for an amino acid. The products of multiple GCD genes are required for efficient repression of GCN4 mRNA translation under nonstarvation conditions. The majority of the known GCD genes encode subunits of the general translation initiation factor eIF-2 or eIF-2B. To identify additional initiation factors in yeast, we characterized 65 spontaneously arising Gcd− mutants. In addition to the mutations that were complemented by known GCD genes or by GCN3, we isolated mutant alleles of two new genes named GCD14 and GCD15. Recessive mutations in these two genes led to highly unregulated GCN4 expression and to derepressed transcription of genes in the histidine biosynthetic pathway under GCN4 control. The derepression of GCN4 expression in gcd14 and gcd15 mutants occurred with little or no increase in GCN4 mRNA levels, and it was dependent on upstream open reading frames (uORFs) in GCN4 mRNA that regulate its translation. We conclude that GCD14 and GCD15 are required for repression of GCN4 mRNA translation by the uORFs under conditions of amino acid sufficiency. The gcd14 and gcd15 mutations confer a slow-growth phenotype on nutrient-rich medium, and gcd15 mutations are lethal when combined with a mutation in gcd13. Like other known GCD genes, GCD14 and GCD15 are therefore probably required for general translation initiation in addition to their roles in GCN4-specific translational control.

scholarly journals Mutual Cross Talk between the Regulators Hac1 of the Unfolded Protein Response and Gcn4 of the General Amino Acid Control of Saccharomyces cerevisiae

2013 ◽  
Vol 12 (8) ◽  
pp. 1142-1154 ◽  
Author(s):  
Britta Herzog ◽  
Blagovesta Popova ◽  
Antonia Jakobshagen ◽  
Hedieh Shahpasandzadeh ◽  
Gerhard H. Braus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Cross Talk ◽  
Cellular Response ◽  
Promoter Regions ◽  
Content Type ◽  
General Amino Acid Control ◽  
Acid Control ◽  
The Unfolded Protein Response ◽  
Diploid Cells

ABSTRACTHac1 is the activator of the cellular response to the accumulation of unfolded proteins in the endoplasmic reticulum. Hac1 function requires the activity of Gcn4, which mainly acts as a regulator of the general amino acid control network providingSaccharomyces cerevisiaecells with amino acids. Here, we demonstrate novel functions of Hac1 and describe a mutual connection between Hac1 and Gcn4. Hac1 is required for induction of Gcn4-responsive promoter elements in haploid as well as diploid cells and therefore participates in the cellular amino acid supply. Furthermore, Hac1 and Gcn4 mutually influence their mRNA expression levels. Hac1 is also involved inFLO11expression and adhesion upon amino acid starvation. Hac1 and Gcn4 act through the same promoter regions of theFLO11flocculin. The results indicate an indirect effect of both transcription factors onFLO11expression. Our data suggest a complex mutual cross talk between the Hac1- and Gcn4-controlled networks.

The Saccharomyces cerevisiae ADE1 gene: structure, overexpression and possible regulation by general amino acid control

Gene ◽  
1991 ◽  
Vol 109 (1) ◽  
pp. 143-147 ◽  
Author(s):  
Andrey N. Myasnikov ◽  
Kestutis V. Sasnauskas ◽  
Arvidas A. Janulaitis ◽  
Mikhail N. Smirnov
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Gene Structure ◽  
General Amino Acid Control ◽  
Acid Control

scholarly journals Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis.

1989 ◽  
Vol 9 (7) ◽  
pp. 3101-3104 ◽  
Author(s):  
G M Cole ◽  
D Schild ◽  
R K Mortimer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Meiotic Recombination ◽  
Gene Fusion ◽  
Lacz Gene ◽  
Alpha Cells ◽  
Galactosidase Activity ◽  
A Cells ◽  
Beta Galactosidase

The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/alpha cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of beta-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

scholarly journals Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (11) ◽  
pp. 2347-2355 ◽  
Author(s):  
S D Emr ◽  
I Schauer ◽  
W Hansen ◽  
P Esmon ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Lacz Gene ◽  
Gene Fusions ◽  
Glucose Medium ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Hybrid Proteins ◽  
Suc2 Gene ◽  
Beta Galactosidase

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.

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