scholarly journals The translational activator GCN3 functions downstream from GCN1 and GCN2 in the regulatory pathway that couples GCN4 expression to amino acid availability in Saccharomyces cerevisiae.

Genetics ◽  
1990 ◽  
Vol 126 (3) ◽  
pp. 549-562 ◽  
Author(s):  
E M Hannig ◽  
N P Williams ◽  
R C Wek ◽  
A G Hinnebusch
Keyword(s):  
Amino Acid ◽  
State Level ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Temperature Sensitive ◽  
General Control ◽  
Biosynthetic Genes ◽  
Protein Coding ◽  
General Amino Acid Control ◽  
Amino Acid Availability

Abstract The GCN4 protein of S. cerevisiae is a transcriptional activator of amino acid biosynthetic genes which are subject to general amino acid control. GCN3, a positive regulator required for increased GCN4 expression in amino acid-starved cells, is thought to function by antagonism of one or more negative regulators encoded by GCD genes. We isolated gcn3c alleles that lead to constitutively derepressed expression of GCN4 and amino acid biosynthetic genes under its control. These mutations map in the protein-coding sequences and, with only one exception, do not increase the steady-state level of GCN3 protein. All of the gcn3c alleles lead to derepression of genes under the general control in the absence of GCN1 and GCN2, two other positive regulators of GCN4 expression. This finding suggests that GCN3 functions downstream from GCN1 and GCN2 in the general control pathway. In accord with this idea, constitutively derepressing alleles of GCN2 are greatly dependent on GCN3 for their derepressed phenotype. The gcn3c alleles that are least dependent on GCN1 and GCN2 for derepression cause slow-growth under nonstarvation conditions. In addition, all of the gcn3c alleles are less effective than wild-type GCN3 in overcoming the temperature-sensitive lethality associated with certain mutations in the negative regulator GCD2. These results suggest that activation of GCN3 positive regulatory function by the gcn3c mutations involves constitutive antagonism of GCD2 function, leading to reduced growth rates and derepression of GCN4 expression in the absence of amino acid starvation.

scholarly journals Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (9) ◽  
pp. 3150-3155 ◽  
Author(s):  
P L Myers ◽  
R C Skvirsky ◽  
M L Greenberg ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Regulatory Gene ◽  
Negative Regulator ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
General Control ◽  
Biosynthetic Genes ◽  
Isolation And Characterization ◽  
Steady State Levels

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.

scholarly journals gcd12 mutations are gcn3-dependent alleles of GCD2, a negative regulator of GCN4 in the general amino acid control of Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 543-550 ◽  
Author(s):  
C J Paddon ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Growth Defect ◽  
Gene Products ◽  
Wild Type ◽  
General Amino Acid Control ◽  
Translational Repressor ◽  
Acid Control

Abstract GCD12 encodes a translational repressor of the GCN4 protein, a transcriptional activator of amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. gcd12 mutations override the requirement for the GCN2 and GCN3 gene products for derepression of GCN4 expression, suggesting that GCN2 and GCN3 function indirectly as positive regulators by negative regulation of GCD12. In addition to their regulatory phenotype, gcd12 mutants are temperature-sensitive for growth (Tsm-) and, as shown here, deletion of the GCD12 gene is unconditionally lethal. Both the regulatory and the Tsm- phenotypes associated with gcd12 point mutations are completely overcome by wild-type GCN3, implying that GCN3 can promote or partially substitute for the functions of GCD12 in normal growth conditions even though it antagonizes GCD12 regulatory function in starvation conditions. The GCD12 gene has been cloned and mapped to the right arm of chromosome VII, very close to the map position reported for GCD2. We demonstrate that GCD12 and GCD2 are the same genes; however, unlike gcd12 mutations, the growth defect and constitutive derepression phenotypes associated with the gcd2-1 mutation are expressed in the presence of the wild-type GCN3 gene. These findings can be explained by either of two alternative hypotheses: (1) gcd12 mutations affect a domain of the GCD2 protein that directly interacts with GCN3, and complex formation stabilizes mutant gcd12 (but not gcd2-1) gene products; (2) gcd12 mutations selectively impair one function of GCD2 that is replaceable by GCN3, whereas gcd2-1 inactivates a different GCD2 function for which GCN3 cannot substitute. Both models imply a close interaction between these two positive and negative regulators in general amino acid control.

Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (9) ◽  
pp. 3150-3155
Author(s):  
P L Myers ◽  
R C Skvirsky ◽  
M L Greenberg ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Regulatory Gene ◽  
Negative Regulator ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
General Control ◽  
Biosynthetic Genes ◽  
Isolation And Characterization ◽  
Steady State Levels

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.

scholarly journals Amino acid deprivation induces AKT activation by inducing GCN2/ATF4/REDD1 axis

2021 ◽  
Vol 12 (12) ◽  
Author(s):  
Hyeon-Ok Jin ◽  
Sung-Eun Hong ◽  
Ji-Young Kim ◽  
Se-Kyeong Jang ◽  
In-Chul Park
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Survival ◽  
Amino Acid Deprivation ◽  
General Control ◽  
Akt Activation ◽  
Amino Acid Availability ◽  
Survival Signal ◽  
Cancer Cell Survival ◽  
Glutamine Deprivation

AbstractAmino acid availability is sensed by various signaling molecules, including general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). However, it is unclear how these sensors are associated with cancer cell survival under low amino acid availability. In the present study, we investigated AKT activation in non-small cell lung cancer (NSCLC) cells deprived of each one of 20 amino acids. Among the 20 amino acids, deprivation of glutamine, arginine, methionine, and lysine induced AKT activation. AKT activation was induced by GCN2/ATF4/REDD1 axis-mediated mTORC2 activation under amino acid deprivation. In CRISPR-Cas9-mediated REDD1-knockout cells, AKT activation was not induced by amino acid deprivation, indicating that REDD1 plays a major role in AKT activation under amino acid deprivation. Knockout of REDD1 sensitized cells cultured under glutamine deprivation conditions to radiotherapy. Taken together, GCN2/ATF4/REDD1 axis induced by amino acid deprivation promotes cell survival signal, which might be a potential target for cancer therapy.

Molecular analysis of GCN3, a translational activator of GCN4: evidence for posttranslational control of GCN3 regulatory function

1988 ◽  
Vol 8 (11) ◽  
pp. 4808-4820
Author(s):  
E M Hannig ◽  
A G Hinnebusch
Keyword(s):  
Amino Acid ◽  
Normal Growth ◽  
Growth Conditions ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Noncoding Dna ◽  
Physical Description ◽  
Amino Acid Availability ◽  
Translational Activator ◽  
Starvation Conditions

GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. The GCN3 product is a positive regulator required for increased synthesis of GCN4 protein in amino acid-starved cells. GCN3 appears to act indirectly by antagonizing GCD-encoded negative regulators of GCN4 expression under starvation conditions; however, GCN3 can also suppress the effects of gcd12 mutations under nonstarvation conditions. These results imply that the GCN3 product can promote either repression or activation of GCN4 expression depending on amino acid availability. We present a complete physical description of the GCN3 gene and its transcript, plus measurements of GCN3 expression at the transcriptional and translational levels under different growth conditions. GCN3 encodes a 305-amino-acid polypeptide with no significant homology to any other known protein sequence. GCN3 mRNA contains no leader AUG codons, and no potential GCN4 binding sites were found in GCN3 5' noncoding DNA. In accord with the absence of these regulatory sequences found at other genes in the general control system, GCN3 mRNA and a GCN3-lacZ fusion enzyme are present at similar levels under both starvation and nonstarvation conditions. These data suggest that modulation of GCN3 regulatory function in response to amino acid availability occurs posttranslationally. A gcn3 deletion leads to unconditional lethality in a gcd1-101 mutant, supporting the idea that GCN3 is expressed under normal growth conditions and cooperates with the GCD1 product under these circumstances to carry out an essential cellular function. We describe a point mutation that adds three amino acids to the carboxyl terminus of GCN3, which inactivates its positive regulatory function required under starvation conditions without impairing its ability to promote functions carried out by GCD12 under nonstarvation conditions.

scholarly journals Regulation of type I collagen mRNA in lung fibroblasts by cystine availability

1998 ◽  
Vol 331 (2) ◽  
pp. 417-422 ◽  
Author(s):  
David C. RISHIKOF ◽  
Ping-Ping KUANG ◽  
Christine POLIKS ◽  
Ronald H. GOLDSTEIN
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Amino Acid ◽  
Type I Collagen ◽  
State Level ◽  
Lung Fibroblasts ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Mrna ◽  
Amino Acid Availability

The steady-state level of α1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases α1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of α1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of α1(I) collagen mRNA. We found that re-expression of α1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of α1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased α1(I) collagen mRNA levels. The combination of glutamine and cystine increased α1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase α1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of α1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of γ-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of α1(I) collagen mRNA.

scholarly journals Molecular analysis of GCN3, a translational activator of GCN4: evidence for posttranslational control of GCN3 regulatory function.

1988 ◽  
Vol 8 (11) ◽  
pp. 4808-4820 ◽  
Author(s):  
E M Hannig ◽  
A G Hinnebusch
Keyword(s):  
Amino Acid ◽  
Normal Growth ◽  
Growth Conditions ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Noncoding Dna ◽  
Physical Description ◽  
Amino Acid Availability ◽  
Translational Activator ◽  
Starvation Conditions

GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. The GCN3 product is a positive regulator required for increased synthesis of GCN4 protein in amino acid-starved cells. GCN3 appears to act indirectly by antagonizing GCD-encoded negative regulators of GCN4 expression under starvation conditions; however, GCN3 can also suppress the effects of gcd12 mutations under nonstarvation conditions. These results imply that the GCN3 product can promote either repression or activation of GCN4 expression depending on amino acid availability. We present a complete physical description of the GCN3 gene and its transcript, plus measurements of GCN3 expression at the transcriptional and translational levels under different growth conditions. GCN3 encodes a 305-amino-acid polypeptide with no significant homology to any other known protein sequence. GCN3 mRNA contains no leader AUG codons, and no potential GCN4 binding sites were found in GCN3 5' noncoding DNA. In accord with the absence of these regulatory sequences found at other genes in the general control system, GCN3 mRNA and a GCN3-lacZ fusion enzyme are present at similar levels under both starvation and nonstarvation conditions. These data suggest that modulation of GCN3 regulatory function in response to amino acid availability occurs posttranslationally. A gcn3 deletion leads to unconditional lethality in a gcd1-101 mutant, supporting the idea that GCN3 is expressed under normal growth conditions and cooperates with the GCD1 product under these circumstances to carry out an essential cellular function. We describe a point mutation that adds three amino acids to the carboxyl terminus of GCN3, which inactivates its positive regulatory function required under starvation conditions without impairing its ability to promote functions carried out by GCD12 under nonstarvation conditions.

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.

New positive and negative regulators for general control of amino acid biosynthesis in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1820-1829
Author(s):  
M L Greenberg ◽  
P L Myers ◽  
R C Skvirsky ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Regulatory Genes ◽  
Negative Regulator ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
General Control ◽  
Positive Regulators ◽  
Direct Acting ◽  
Analog Sensitivity

The biosynthesis of most amino acids in Saccharomyces cerevisiae is coregulated. Starvation for a single amino acid results in the derepression of amino acid biosynthetic enzymes in many unrelated pathways. This phenomenon, known as general control, is mediated by both positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the identification and characterization of several new regulatory genes for this system, GCN6, GCN7, GCN8, GCN9, and GCD5. A mutation in the negative regulator GCD5 was isolated on the basis of its suppression of a gcn2 mutation. The effect of gcd5 is a posttranscriptional increase in histidine biosynthetic enzyme activity. Suppressors of gcd5 which are deficient in derepression were in turn isolated. Eight such mutations, defining four new positive regulatory genes (GCN6 through GCN9), were obtained. These mutations are recessive, confer sensitivity to multiple amino acid analogs, and result in decreased mRNA levels for genes under general control. The GCN6 and GCN7 gene products were shown to be positive regulators for transcription of the GCN4 gene, the most direct-acting positive regulator thus far identified. The interaction of GCN6 and GCN7 with GCN4 is fundamentally different from that of previously isolated GCN genes. It should also be noted that these gcn selections gave a completely different nonoverlapping set of mutations from earlier selections which relied on analog sensitivity. Thus, we may have identified a new class of GCN genes which are functionally distinct from GCN1 through GCN5.

scholarly journals The general amino acid control pathway regulates mTOR and autophagy during serum/glutamine starvation

2014 ◽  
Vol 206 (2) ◽  
pp. 173-182 ◽  
Author(s):  
Rui Chen ◽  
Yilong Zou ◽  
Dongxue Mao ◽  
Daxiao Sun ◽  
Guanguang Gao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Amino Acid Level ◽  
Amino Acid Uptake ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
General Amino Acid Control ◽  
Amino Acid Availability ◽  
Acid Control ◽  
Activating Transcription Factor 4

Organisms have evolved elaborate mechanisms to adjust intracellular nutrient levels in response to fluctuating availability of exogenous nutrients. During starvation, cells can enhance amino acid uptake and synthesis through the general amino acid control (GAAC) pathway, whereas nonessential cellular contents are recycled by autophagy. How these two pathways are coordinated in response to starvation is currently unknown. Here we show that the GAAC pathway couples exogenous amino acid availability with autophagy. Starvation caused deactivation of mTOR, which then activated autophagy. In parallel, serum/glutamine starvation activated the GAAC pathway, which up-regulated amino acid transporters, leading to increased amino acid uptake. This elevated the intracellular amino acid level, which in turn reactivated mTOR and suppressed autophagy. Knockdown of activating transcription factor 4, the major transcription factor in the GAAC pathway, or of SLC7A5, a leucine transporter, caused impaired mTOR reactivation and much higher levels of autophagy. Thus, the GAAC pathway modulates autophagy by regulating amino acid uptake and mTOR reactivation during serum/glutamine starvation.

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