Changes in gene expression elicited by amino acid limitation in Neurospora crassa strains having normal or mutant cross-pathway amino acid control

Summary The effects of amino acid limitation on gene expression have been investigated in Neurospora crassa strains carrying normal (cpc-1 ⁺) or mutant (cpc-1) alleles at a locus implicated in cross-pathway amino acid control. Electrophoresis and fluorography were used to reveal the patterns of label incorporation into polypeptides in vivo, or after in vitro translation of extracted mRNAs. In a cpc-1 ⁺ strain at least 20% of detectable in vitro translation products showed relative increases in incorporation when RNA was obtained from mycelium grown under conditions of arginine limitation, by comparison with conditions of arginine sufficiency. A cpc-1 mutation, which impairs derepression of a variety of amino acid synthetic enzymes following amino acid limitation, had little detectable effect on in vivo polypeptide synthesis during amino acid sufficient growth or following pyrimidine limitation. However the mutation substantially altered the response to arginine or histidine limitation. The majority of in vitro translation products that showed increased expression in arginine limited cpc-1 ⁺ failed to increase in cpc-1 strains, but arginine limitation of cpc-1 also resulted in increases that did not occur in cpc-1 ⁺ strains. This may reflect both direct and indirect consequences of the impairment of cross-pathway control.     

Cross-pathway control of ornithine carbamoyltransferase synthesis in Neurospora crassa

The pattern of cross-pathway regulation of the arginine synthetic enzyme ornithine carbamoyltransferase was investigated in Neurospora crassa, using single and double mutant auxotrophic strains starved for their required amino acids. These experiments show that starvation for histidine, tryptophan, isoleucine, valine or arginine can result in derepression of ornithine carbamoyltransferase. Methionine starvation also gave slight derepression, but starvation for lysine or leucine gave little or no effect.     

Cross-Pathway Regulation: Histidine-Mediated Control of Histidine, Tryptophan, and Arginine Biosynthetic Enzymes in Neurospora crassa

In Neurospora crassa, histidine starvation of histidine mutants resulted in derepression of histidine, tryptophan, and arginine biosynthetic enzymes. The same tripartite derepression occurred in wild-type strain 74A when it was grown in medium supplemented with 3-amino-1,2,4-triazole, an inhibitor of histidine biosynthesis. Histidine-mediated derepression of tryptophan and arginine biosynthetic enzymes was not due to a lowered intracellular concentration of tryptophan or arginine, respectively. A discussion of possible mechanisms and of similar studies in prokaryotic and eukaryotic organisms is presented.     

Cross-Pathway Regulation: Tryptophan-Mediated Control of Histidine and Arginine Biosynthetic Enzymes in Neurospora crassa

In Neurospora crassa, the starvation of tryptophan mutants for tryptophan resulted in the derepression of tryptophan, histidine, and arginine biosynthetic enzymes. This tryptophan-mediated derepression of histidine and arginine biosynthetic enzymes occurred despite the fact that the tryptophan-starved cells had a higher intracellular concentration of histidine and arginine than did nonstarved cells.     

Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids

Summary Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of l-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. A cpc-1 mutant strain, impaired in cross-pathway regulation i.e. lacking the ability to derepress, shows delayed growth under such conditions. In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all. Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.     

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

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.     

Tryptophan biosynthesis in Saccharomyces cerevisiae: control of the flux through the pathway.

Enzyme derepression and feedback inhibition of the first enzyme are the regulatory mechanisms demonstrated for the tryptophan pathway in Saccharomyces cerevisiae. The relative contributions of the two mechanisms to the control of the flux through the pathway in vivo were analyzed by (i) measuring feedback inhibition of anthranilate synthase in vivo, (ii) determining the effect of regulatory mutations on the level of the tryptophan pool and the flux through the pathway, and (iii) varying the gene dose of individual enzymes of the pathway at the tetraploid level. We conclude that the flux through the pathway is adjusted to the rate of protein synthesis by means of feedback inhibition of the first enzyme by the end product, tryptophan. The synthesis of the tryptophan enzymes could not be repressed below a basal level by tryptophan supplementation of the media. The enzymes are present in excess. Increasing or lowering the concentration of individual enzymes had no noticeable influencing on the overall flux to tryptophan. The uninhibited capacity of the pathway could be observed both upon relieving feedback inhibition by tryptophan limitation and in feedback-insensitive mutants. It exceeded the rate of consumption of the amino acid on minimal medium by a factor of three. Tryptophan limitation caused derepression of four of the five tryptophan enzymes and, as a consequence, led to a further increase in the capacity of the pathway. However, because of the large reserve capacity of the "repressed" pathway, tryptophan limitation could not be imposed on wild-type cells without resorting to the use of analogs. Our results, therefore, suggest that derepression does not serve as an instrument for the specific regulation of the flux through the tryptophan pathway.     

Control of arginine utilization in Neurospora.

The response of Neurospora to changes in the availibility of exogenous arginine was investigated. Upon addition of arginine to the growth medium, catabolism is initiated within minutes. This occurs prior to expansion of the arginine pool or augmentation of catabolic enzyme levels. (Basal levels are approximately 25% of those found during growth in arginine-supplemented medium.) Catabolism of arginine is independent of protein synthesis, indicating that the catabolic enzymes are active but that arginine is not available for catabolism unless present in the medium. Upon exhaustion of the supply of exogenous arginine, catabolism ceases abruptly, despite an expanded arginine pool and induced levels of the catabolic enzymes. The arginine pool supports protein synthesis until the cells regain their normal capacity for endogenous arginine synthesis. These observations, combined with the known small level of induction of arginine catabolic enzymes, non-repressibility of most biosynthetic enzymes, and vesicular localization of the bulk of the arginine pool, suggest that compartmentation plays a significant role in controlling arginine metabolism in Neurospora.     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Mobilization of vacuolar arginine in Neurospora crassa. Mechanism and role of glutamine

Nitrogen starvation has been shown to increase the cytosolic arginine concentration and to accelerate protein turnover in mycelia of Neurospora crassa. The cytosolic arginine is derived from a metabolically inactive vacuolar pool. Redistribution of arginine between cytosolic and vacuolar compartments is the result of mobilization of this metabolite in response to nitrogen starvation. Mobilization of arginine (and purines) also occurred in response to glutamine limitation, but arginine accumulated upon proline starvation. These observations indicate that mobilization is a consequence of glutamine limitation rather than a general response to amino acid starvation (or limitation). Analysis of the amino acid pools in mycelia subjected to starvation or limitation suggests that glutamine (or a metabolite derived from glutamine) provides a signal which determines the metabolic fate of vacuolar arginine. The results are consistent with the hypothesis that vacuolar compartmentation provides a readily available store of nitrogen-rich compounds to be utilized during differentiation or under conditions of nutritional stress.     

Three regulatory systems control production of glutamine synthetase in Saccharomyces cerevisiae.

Production of glutamine synthetase in Saccharomyces cerevisiae is controlled by three regulatory systems. One system responds to glutamine levels and depends on the positively acting GLN3 product. This system mediates derepression of glutamine synthetase in response to pyrimidine limitation as well, but genetic evidence argues that this is an indirect effect of depletion of the glutamine pool. The second system is general amino acid control, which couples derepression of a variety of biosynthetic enzymes to starvation for many single amino acids. This system operates through the positive regulatory element GCN4. Expression of histidinol dehydrogenase, which is under general control, is not stimulated by glutamine limitation. A third system responds to purine limitation. No specific regulatory element has been identified, but depression of glutamine synthetase is observed during purine starvation in gln3 gcn4 double mutants. This demonstrates that a separate purine regulatory element must exist. Pulse-labeling and immunoprecipitation experiments indicate that all three systems control glutamine synthetase at the level of subunit synthesis.     

Effect of chloramphenicol and ethidium bromide on the level of ornithine carbamoyltransferase in Neurospora crassa.

The specific activity of the nuclear-gene-encoded, mitochondrial arginine biosynthetic enzyme ornithine carbamoyltransferase (EC 2.1.3.3) in Neurospora crassa was elevated in mycelia treated with chloramphenicol or ethidium bromide. The increase in specific activity was caused by an increase in the number of mature enzyme molecules rather than by the activation of a preexisting enzyme. Chloramphenicol and ethidium bromide appeared to act indirectly via arginine-mediated derepression. However, derepression did not appear to result from a drug-mediated decrease in the arginine pool.     

Histidine-Mediated Control of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of the five tryptophan biosynthetic enzymes of Neurospora crassa was shown to be derepressed in histidine-starved cells. This histidine-mediated derepression was not due to a lowered intracellular concentration of tryptophan in these cells. Furthermore, histidine-mediated derepression of tryptophan enzymes was found to be coordinate and not subject to reversal by tryptophan of either exogenous or biosynthetic origin. The synthesis of tryptophan enzymes also was found to be coordinate in cells which were not histidine-starved. Although histidine is clearly involved in regulating the synthesis of tryptophan enzymes, it did not prevent either tryptophan-mediated derepression of tryptophan enzymes or indole-3-glycerol phosphate-mediated derepression of tryptophan synthetase.