Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.     

Induction of the allantoin degradative enzymes by allophanic acid, the last intermediate of the pathway

$\text{Saccharomyces cerevisiae}$ can utilize allantoin as a sole nitrogen source by degrading it in five steps to ammonia, “CO₂”, and glyoxylate. We have previously shown that allophanic acid is the inducer of the urea carboxylase: allophanate hydrolase multienzyme complex. Since these enzymes catalyse the last two steps of allantoin degradation, experiments were performed to determine if allophanate was also the inducer of any other enzymes in the pathway. Our data demonstrate that allophanate induces synthesis of at least five of the seven purine degradative enzymes.     

Pleiotropic control of five eucaryotic genes by multiple regulatory elements.

We have previously shown that allophanate acts as an inducer for five structural genes whose products participate in the degradation of allantoin by Saccharomyces cerevisiae. This observation led us to hypothesize that these genes might be controlled in common and to test the hypothesis by searching for mutants unable to induce production of the allantoin-degrading enzymes. Such mutants have been found. These strains grew poorly when provided with any of the allantoin pathway intermediates, but used other nitrogen sources normally. The mutations carried in these strains were recessive to wild-type alleles and complemented mutations in all known loci associated with the allantoin pathway. The locus containing the most thoroughly studied mutation (dal81-1) was not fund to be tightly linked to any of the allantoin pathway structural genes. The low basal levels of allantoin pathway enzymes observed in Dal81- strains remained the same whether or not the inducer was present in the growth medium. However, the levels of enzyme increased moderately when mutants were grown on poor nitrogen sources. From these observations, we conclude that dal81 mutant strains possess a defect in the induction of enzyme synthesis; enzyme production due to relief of nitrogen catabolite repression, however, appears normal. The observed epistatic relationships of mutations in the DAL80 and DAL81 loci suggest that their products may possess a reasonable degree of functional independence.     

Oxaluric Acid: a Non-Metabolizable Inducer of the Allantoin Degradative Enzymes in Saccharomyces cerevisiae

Saccharomyces cerevisiae degrades allantoin in five steps to ammonia, CO(2), and glyoxylate. Previously we demonstrated that allophanic acid, the last intermediate of the pathway, was required for induction of all five degradative enzymes. The data presented here indicate that oxaluric acid, an allophanate analogue, is capable of serving as a non-metabolizable inducer. Oxaluric acid brings about a high level of induction even in strains lacking urea carboxylase. Induction observed with parabanic acid was found to result from its spontaneous breakdown to oxaluric acid.     

Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae.

Saccharomyces cerevisiae responds to deperivation of nutrients by arresting cell division at the unbudded G1 stage. Cells situated outside of G1 at the time of deperivation complete the cell cycle before arresting. This prompted an investigation of the source of nutrients used by these cells to complete division and the mechanisms controlling their availability. We found a close correlation between accumulation of unbudded cells and loss of previously formed allophanate hydrolase activity after nutrient starvation. These losses were not specific to the allantoin, system since they have been observed for a number of other enzymes and also when cellular protein levels were monitored with [3H]leucine. Loss of hydrolase activity was also observed when protein synthesis was inhibited either by addition of inhibitors or loss of the prtl gene product. We found that onset of nutrient starvation brought about release of large quantities of arginine and allantoin normally sequestered in the cell vacuole. Treatment of a cells with alpha-factor resulted in both the release of allantoin and arginine from the cell vacuole and the onset of intracellular protein degradation. These effects were not observed when either alpha cells or a/alpha diploid strains were treated with alpha-factor. These data suggest that release of vacuolar constitutents and protein turnover may be regulated by the G1 arrest signal.     

Oxalurate transport in Saccharomyces cerevisiae.

Oxalurate, the gratuitous inducer of the allantoin degradative enzymes, was taken into the cell by an energy-dependent active transport system with an apparent Km of 1.2 mM. Efflux of previously accumulated oxalurate was rapid, with a half-life of about 2 min. The oxalurate uptake system appears to be both constitutively produced and insensitive to nitrogen catabolite repression. The latter observations suggest that failure of oxalurate to bring about induction of allophanate hydrolase in cultures growing under repressive conditions does not result from inducer exclusion, but rather from repression of dur1,2 gene expression.     

Metabolic control of urea catabolism in Chlamydomonas reinhardi and Chlorella pyrenoidosa.

In the unicellular green alga Chlamydomonas reinhardi (strain y-1), synthesis of the enzymes required for urea hydrolysis is under substrate induction control by urea and under end product repression control by ammonia. Hydrolysis of urea if effected by the sequential action of the discrete enzymes urea carboxylase and allophanate lyase, collectively called urea amidolyase. The carboxylase converts urea to allophanate in a reaction requiring biotin, adenosine 5'-triphosphate, and Mg2+. The lyase hydrolzyes allophanate to ammonium ions and bicarbonate. Neither activity is present in more than trace amounts when cultures are grown with ammonia or urea plus ammonia, or when they are starved for nitrogen for 8 h. Urea in the absence of ammonia induces both activities 10 to 100 times the basal levels. Addition of ammonia to an induced culture causes complete cessation of carboxylase accumulation and an 80% depression of lyase accumulation. Ammonia does not reduce urea uptake by repressed cells, so it does not prevent induction by the mechanism of inducer exclusion. The unicellular green alga Chlorella pyrenoidosa (strain 3 Emerson) also has discrete carboxylase and lyase enzymes, but only the carboxylase exhibits metabolic control.     

Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.

Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein.     

Urea formation in the green vegetable bug Nezara viridula

Urea comprises 7·7 per cent of the total nitrogen excretion of Nezara viridula. The bug is capable of oxidizing uric acid to allantoin, which is also excreted, but the uricolytic pathway is not active beyond this point. Of the enzymes of the ornithine cycle, arginase and ornithine transcarbamalase are active, but there is no evidence for the arginine synthetase system. Carbamyl phosphate synthetase has a low activity detectable only by the use of radioactive substrates. Confirmation of the operation of only part of the ornithine cycle is seen in the incorporation of bicarbonate carbon into citrulline, but not into arginine or urea, by homogenates of bug tissue. It is concluded that urea in the excreta is derived from excess arginine in the diet by the action of the enzyme arginase. Free arginine is present in the cell sap of the bean pods on which the bugs feed in amounts sufficient to account for the urea excreted.     

Induction and derepression of arginase and ornithine transaminase in different strains ofSaccharomyces cerevisiae

The syntheses of arginase and ornithine transaminase were studied in two strains ofSaccharomyces cerevisiae, viz. strain B and strain α-Σ1278b. Derepression of both enzymes during nitrogen starvation was shown only by strain B, non-specific induction of arginase only by strain α-Σ1278b. This different response of both strains studied reveals substantial differences in the regulation of enzyme synthesis among yeast strains of one and the same species. The specific enzyme activities observed in chemostat cultures with arginine as the nitrogen source and different sugars, at variable carbon to nitrogen ratios, did not indicate the involvement of carbon catabolite repression in the regulation of arginase and ornithine transaminase syntheses. Specific arginase activities observed in the continuous cultures varied widely and did not show a correlation with the intracellular arginine concentration. Extracellular steady-state arginine concentrations higher than about 0.1mm, in addition to abundant energy supply, were found to be required for high production of arginase. It is suggested that, besides intracellular arginine, extracellular arginine may provide an induction signal necessary for full-scale induction of arginase synthesis. A possible intermediary role of arginine permeases or of other membrane proteins is discussed.     

Transcriptional regulation of the DAL5 gene in Saccharomyces cerevisiae

We demonstrate that the DAL5 gene, encoding a necessary component of the allantoate transport system, is constitutively expressed in Saccharomyces cerevisiae. Its relatively high basal level of expression did not increase further upon addition of allantoin pathway intermediates. However, steady-state DAL5 mRNA levels dropped precipitously when a repressive nitrogen source was provided. These control characteristics of DAL5 expression make this gene a good model with which to unravel the mechanism of nitrogen catabolite repression. Its particular advantage relative to other potentially useful genes derives from its lack of control by induction and hence the complicating effects of inducer exclusion.     

Effect of Carbon Source on Enzymes and Metabolites of Arginine Metabolism in Neurospora

The levels of enzymes and metabolites of arginine metabolism were determined in exponential cultures of Neurospora crassa grown on various carbon sources. The carbon sources decreased in effectiveness (as determined by generation times) in the following order: sucrose, acetate, glycerol, and ethanol. The basal and induced levels of the catabolic enzymes, arginase (EC 3.5.3.1) and ornithine transaminase (EC 2.6.1.13), were lower in mycelia grown on poor carbon sources. Arginase was more sensitive to variations in carbon source than was ornithine transaminase. Induction of both enzymes was sensitive to nitrogen metabolite control, but this sensitivity was reduced in mycelia grown on glycerol or ethanol. The pools of arginine and ornithine were reduced in mycelia grown in unsupplemented medium containing poor carbon sources, but the biosynthetic enzyme ornithine transcarbamylase (EC 2.1.3.3) was not derepressed. The arginine pools were similar, regardless of carbon source, in mycelia grown in arginine-supplemented medium. The ornithine pool was reduced by growth on poor carbon sources. The rate of arginine degradation was proportional to the level of arginase in both sucrose- and glycerol-grown mycelia. The distribution of arginine between cytosol and vesicles was only slightly altered by growth on glycerol instead of sucrose. The slightly smaller cytosolic arginine concentration did not appear to be sufficient to account for the alterations in basal and induced enzyme levels. The results suggest a possible carbon metabolite effect on the expression or turnover of a variety of genes for enzymes of arginine metabolism in Neurospora.     

Effect of Dietary Addition of Amino Acids and Proteins on the Activities of Some Urea Cycle Enzymes in Rat Liver

The activities of ornithine transcarbamylase, arginine synthetase and arginase in the liver of rats receiving basal diets containing 25% casein supplemented respectively with arginine, aspartic acid, glutamic acid, glycine, a mixture of arginine, aspartic acid and glutamic acid, egg albumin, casein, wheat gluten and gelatin have been determined.

These urea cycle enzymes in rats receiving diets supplemented with the various nitrogen sources were generally increased, but the increments were due to the increase of the ingested amount of nitrogen, and not the specific effect of the individual amino acids or proteins. The excretion of urinary urea in general was increased proportionally with the elevations of these enzyme activities, independent of the nature of the dietary nitrogen.     

Identification of the ureidoglycolate hydrolase gene in the DAL gene cluster of Saccharomyces cerevisiae.

This report describes the isolation of the genes encoding allantoicase (DAL2) and ureidoglycolate hydrolase (DAL3), which are components of the large DAL gene cluster on the right arm of chromosome IX of Saccharomyces cerevisiae. During this work a new gene (DAL7) was identified and found to be regulated in the manner expected for an allantoin pathway gene. Its expression was (i) induced by allophanate, (ii) sensitive to nitrogen catabolite repression, and (iii) responsive to mutation of the DAL80 and DAL81 loci, which have previously been shown to regulate the allantoin degradation system. Hybridization probes generated from these cloned genes were used to analyze expression of the allantoin pathway genes in wild-type and mutant cells grown under a variety of physiological conditions. When comparison was possible, the patterns of mRNA and enzyme levels observed in various strains and physiological conditions were very similar, suggesting that the system is predominantly regulated at the level of gene expression. Although all of the genes seem to be controlled by a common mechanism, their detailed patterns of expression were, at the same time, highly individual and diverse.     

The regulation of urea-biosynthesis enzymes in vertebrates

1. Carbamoyl phosphate synthetase, ornithine transcarbamoylase, the arginine-synthetase system and arginase were measured in the livers of ammoniotelic, ureotelic and uricotelic animals. The chelonian reptiles, whose nitrogen excretory patterns vary according to the habitat, and the Mexican axolotl, a neotenic species, were also studied. 2. The levels of the activities of the first three enzymes mentioned correlate with the amount of nitrogen excreted as urea. 3. The terrestrial turtle, which excretes mainly uric acid, maintains a high arginase activity but has very low levels of the activities of the other three enzymes. 4. The first three enzymes of the urea cycle vary in the phylogenic scale in a co-ordinated manner, which suggests that they are under the same regulatory mechanism. 5. Urea formation from endogenous arginine in vitro has a low efficiency in the Mexican axolotl. 6. The induction of metamorphosis in the Mexican axolotl by the administration of l-tri-iodothyronine, which causes a shift from ammonio-ureotelism to complete ureotelism, is accompanied by an increase mainly in carbamoyl phosphate synthetase and also by an improvement in the efficiency of hydrolysis of endogenous arginine in vitro to give urea. 7. The results obtained by differential centrifugation of the urea-cycle enzymes in rat and Mexican-axolotl livers are presented. The location requirements for the integration of a metabolic cycle are discussed.     

Nitrogen catabolite repression of arginase (CAR1) expression in Saccharomyces cerevisiae is derived from regulated inducer exclusion.

Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.     

Induction and inhibition of the allantoin permease in Saccharomyces cerevisiae.

Allantoin uptake in Saccharomyces cerevisiae is mediated by an energy-dependent, low-Km, active transport system. However, there is at present little information concerning its regulation. In view of this, we investigated the control of alloantoin transport and found that it was regulated quite differently from the other pathway components. Preincubation of appropriate mutant cultures with purified allantoate (commercial preparations contain 17% allantoin), urea, or oxalurate did not significantly increase allantoin uptake. Preincubation with allantoin, however, resulted in a 10- to 15-fold increase in the rate of allantoin accumulation. Two allantoin analogs were also found to elicit dramatic increases in allantoin uptake. Hydantoin and hydantoin acetic acid were able to induce allantoin transport to 63 and 95% of the levels observed with allantoin. Neither of these compounds was able to serve as a sole nitrogen source for S. cerevisiae, and they may be non-metabolizable inducers of the allantoin permease. The rna1 gene product appeared to be required for allantoin permease induction, suggesting that control was exerted at the level of gene expression. In addition, we have shown that allantoin uptake is not unidirectional; efflux merely occurs at a very low rate. Allantoin uptake is also transinhibited by addition of certain amino acids to the culture medium, and several models concerning the operation of such inhibition were discussed.