The induction of urea carboxylase and allophanate hydrolase in Saccharomyces cerevisiae.

Abstract Saccharomyces cerevisiae can utilize urea as a sole nitrogen source; this compound is degraded to CO2 and NH3 by a multienzyme complex (Whitney, P. A., and Cooper, T. G. (1972) Biochem. Biophys. Res. Commun. 49, 45) composed of urea carboxylase and allophanate hydrolase. Since these activities are present in cultures grown in media containing urea, arginine, or a purine base, the nature of the inducer was investigated. The data presented here indicate that it is the intermediate compound, allophanate, which is the inducer of both urea carboxylase and allophanate hydrolase. This conclusion is based on the observation that: (a) urea induces both activities in a wild type strain, (b) urea has no effect on the basal levels of allophanate hydrolase produced by mutants lacking urea carboxylase, and (c) urea has no effect on the high constitutive level of urea carboxylase produced by a mutant lacking allophanate hydrolase. Consistent with this view is the observation that a urea analogue, formamide, cannot induce allophanate hydrolase in a strain lacking urea carboxylase activity, whereas an allophanate analogue, hydantoic acid, is able to serve as an inducer in such a strain. Both of these compounds are able to effect the induction of both activities in a wild type strain. Mutants blocked in either arginase or urea carboxylase were also used to show that the derepression of allophanate hydrolase following the onset of nitrogen starvation is, in fact, internal induction, and is contingent upon the ability of the cells to produce the inducer, allophanate, from internal arginine and urea pools.

Since these activities are present in cultures grown in media containing urea, arginine, or a purine base, the nature of the inducer was investigated.
The data presented here indicate that it is the intermediate compound, allophanate, which is the inducer of both urea carboxylase and allophanate hydrolase. This conclusion is based on the observation that: (a) urea induces both activities in a wild type strain, (b) urea has no effect on the basal levels of allophanate hydrolase produced by mutants lacking urea carboxylase, and (c) urea has no effect on the high constitutive level of urea carboxylase produced by a mutant lacking allophanate hydrolase. Consistent with this view is the observation that a urea analogue, formamide, cannot induce allophanate hydrolase in a strain lacking urea carboxylase activity, whereas an allophanate analogue, hydantoic acid, is able to serve as an inducer in such a strain. Both of these compounds are able to effect the induction of both activities in a wild type strain.
Mutants blocked in either arginase or urea carboxylase were also used to show that the derepressibn of allophanate hydrolase following the onset of nitrogen starvation is, in fact, internal induction, and is contingent upon the ability of the cells to produce the inducer, allophanate, from internal arginine and urea pools.
The yeast, Sacclaaro?lzyces cerevisiae, is capable of using urea as its sole nitrogen SOUI~CC. Whitney aud Cooper (1) have shown that this capability deprllds on the presence of a urea-degradative multienzyme cornlAx This complex is ccmposed of biotin-containing urea carbosylase and allophanate hydrolase catalyzing reactions 1 and 2, respectively (2).
Urea + ATP + HCO,- Allophanate + 2 "CO*" + 2 NH3 (2) Mutants deficient in each of these enzymes have been isolated (2) and the mutant alleles have been shown to be linked.' Thus, the urea-degradative system in yeast appears to possess a biochemical and genetic organization similar to those of a number of biosynthetic pathways containing enzyme aggregates in both yeast and Neurospara (3)(4)(5).
Since it can be shown that the components of the urea-degradative system are organized together both biochemically and genetically, it is reasonable to inquire whether these components are also regulated together. This inquiry was pursued by investigatirlg the nature of the inducer of these two enzymes.
The data presented here show that allophanate, the product of the urea carbosylase Inaction, is the inducer of allophanate hydrolase and, most likely, the inducer of urea carbosylase also. It n-ill be shown that analogues of urea can scr~e as nonmetabolizable inducers of the system only if they can be carbosylated, but that such carbosylation is not required for induction by the nonmetabolizable allophanate analogue, hgdantoic acid. Finally, we shall present results obtained through tlrc use of thrse nonmetabolizable inducers; they indicate that, thr: apparent dcrepression of the urea-degradative system under conditions of nitrogen starvation is contingent upon the presence of the inducer in the ~clls. A preliminary account of this work has alrrady appeared (6).
Culture Conditions-Iii order to ascertain the effect of \-arious physiological situations upon the rate of synthesis of thp urcadegrading enzymes, cells WWP grown iii rninimal medium (2) containing 0.69$ glucose and O.OZo/;, (IYH&SO.+, and the growth was monitored as optical density i n a Klett-Summcrson photo-electric calorimeter. A culture with a density of 100 Klett units contains approximately 2 x lo7 cells per ml. Induction of the urea-degrading enzymes was initiated by addition of urea or one of the analogues at the indicated concentrations.
At appropriate times, thereafter, IO-ml samples of the induced cultures were transferred to a tube containing sufficient cycloheximide to give a final concentration of 10 pg per ml and rapidly cooled to 0". The chilled cells were collected by centrifugation.
After resuspension of the cells in 0.5 ml of 0.05 M Tris, pH 7.9, containing 57, glycerol, 2.0 x low4 M EDTA, and 3.0 x 1OP M mercaptoethanol, the cells were rendered permeable with the method of Ramos et al. (8), with the exception that the procedures were carried out at 0". Assay Conditions-The enzymes of the urea-degradative system were determined using assay conditions similar to those reported earlier (Table I of R.ef. 2). Since it is not presently possible t,o measure urea carboxylase activity in whole cell preparations containing allophanate hydrolase, it was necessary to use the complete, urea amido-lyase, activity as a measure of urea carboxylase activity.
The coupling of the urea carboxylase reaction to that of allophanate hydrolase in wild type cells is reasonable since if the actual in vivo levels of these enzymes are compared under various conditions, a large excess of the hydrolase is always found.
In strains lacking allophanate hydrolase activity, urea carboxylase could be measured directly as the production of [14C]allophanate from [14C]urea. The reaction mixture was identical with that used in the urea amidolyase reaction (Table I of Ref. 2). The reaction was terminated by the addition of an equal volume (I .O ml) of absolute ethanol; the salt concentration was lowered by the addition of 2.0 ml of water and the entire mixture was applied to a Biorex-5 column, 0.5 x 2.5 cm. Urea was eluted by four l.O-ml washes of 1.0 X 10M4 nr Tris buffer, pH 7.9, containing 20 mM NH4HC03.
The allophanate which was adsorbed to the column could then be removed by three l&ml washes with 1.0 x 10m4 M Tris buffer, pH 7.9, containing 0.5 M NH4HC03. Aquasol was added to the eluent and the radioactivity was determined in the scintillation counter.
In a control experiment, 94% of the allophanate was retained by the column and could then be eluted under these conditions. Biorex-5 (an intermediate base resin containing tertiary and quaternary amine groups) was prepared by successive washings with 8 volumes of 1.0 M KHC03 in 0.01 M Tris, pH 7.9, 8' volumes of 1.0 RI NH4HC03 in 0.01 M Tris, pH 7.9, and 8 volumes of 20 mM NH4HC03 in 1.0 X 1OP M Tris, 1rl-i 7.9.

Nature oj Inducer of Urea Carboxylase and Allophanate
Hydrolase-Urea carboxylase and allophanate hydrolase, the two components of the multienzyme complex responsible for the degradation of urea, are found in cells grown in the presence of urea, arginine or allantoir? (9), but not in cells utilizing ammonia as the sole nitrogen source. We have shown that the induction of the urea-degradative syst.em observed following addition of arginine (10) depends upon the degradation of this compound to urea. We, therefore, concluded t'hat the presence of urea was necessary for the induction of urea carboxylase and allophanate hydrolase.
In order to examine this conclusion more carefully, mutant strains were used in conjunction with the wild type to study the induction of urea carboxylase and allophanate hydrolase individually.
The differential rate of synthesis of allophanate hydrolase in 2 12. Lawther and T. G. Cooper, manuscript in preparation.
the presence and absence of added urea is shown in Fig. IA. The addition of urea resulted in about an 8-fold induction over the basal level. When the same experiment was performed with a mutant devoid of urea carboxylase activity (Fig. IB), however, urea was no longer able to serve as an inducer of allophanate hydrolase; in both the presence and absence of urea the mutant cells contained only basal levels of this activity. Urea was also not able to serve as an inducer of allophanate hydrolase in a second independently isolated mutant possessing a defective urea carboxylase.
In a similar type of experiment, urea was used as an inducer of urea carbozylase.
As shown in Fig. 2A, addition of urea to a wild type culture elicited a significant induction of this activity.
However, when a strain lacking allophanate hydrolase was used (Fig. 2B) urea carboxylase was produced at levels similar to those found in induced wild type cultures, whether or not urea was present in the culture medium.
In an analogous set of experiments, each of the three strains just described was grown on minimal medium; at a cell density of 30 Klett units, one culture of each strain received urea, while a second culture received no inducer.
One generation later, at 60 Klett units, all of the cultures were assayed for urea carboxylase and allophanate hydrolase activity. As shown in Table I the amounts of urea carboxylase and allophanate hydrolase in the mutants lacking these activities are significantly below the basal levels; this is consistent with the assumption that these are structural gene mutations.
Here, as in Fig. 1, urea was incapable of eliciting the induction of allophanate hydrolase in a carboxylase minus strain and, in a hydrolase minus strain the level of urea carboxylase activity was independent of added urea (Fig. 2 1. Differential rate of synthesis of allophanate hydrolase in wild type and urea carboxylase-less strains.
The cells were grown to a density of 30 Klett units in minimal medium containing ammonia as the sole nitrogen source; 1.0 X 1OP M urea was added as indicated.
Samoles of the cultures were removed and the levels of allophanate hydrolase were determined as described under "Materials and Methods." The strains used were the wild type, M-25 (A), and the mutant m-hich lacks urea carboxylase activity, M-62 (B). The induction curves were done as described in the legend to Fig. 1 by these data is that allophanate is the inducer of both urea carboxylase and allophanate hydrolase.
In the mutant lacking urea carboxylase activity, urea camlot be converted to allophanate, and therefore, is unable to induce allophanate hydrolase. In the mutant lacking allophanate hydrolase activity, allo- phanate accumulates even in the absence of added urea and illduces urea carboxylase to a high level. This conclusion requires the assumption that cells growing in minimal medium contain an endogenous source of urea; presumably this could be arginine.

Inducers oj Urea-degradative
Enzymes---In order to find a nonmetabolizable inducer of the urea degradation system, the wild type strain was grown on minimal medium with ammonia as sole nitrogen source and several analogucs of urea and allophanate were added to the cultures; after one generation, the cells were harvested and the level of urea amidolyase was determined.
As shown in Table II, urea and the urea analogues formamide, and formyl urea, were able to serve as inducers.
Hydantoic acid, an allophanate analogue, was also an inducer, but a poor one.
The level of induction as a function of inducer concentration was determined for urea, formamide, and hydantoic acid. 111 each case, inducer at the indicated concentration ( Fig. 3) was added to cells growing exponentially on minimal medium and the level of enzyme was determined one generation later. As shown in Fig. 3A, urea is a good inducer and maximal induction was achieved at a concentration of 10P3 hf. It is possible, however, that maximum induction may be achieved at an even lower concentration of urea, because at the lower concentrations of inducer it is likely that the supply of urea in the media is eshausted before the completion of the experiment. The urea analogue, formamide was also a reasonably good inducer (Fig.  3B). The unusual shape of this curve is not understood at this time. One possible explanation is that formamide was not taken up by the cells as well as urea. Hydantoic acid was a relatively poor inducer (Fig. 3C), but it is clear that the addition of this compound did result in an increase in enzyme level.
If the strains used in these studies do not readily take up the formamide and hydantoic acid from the medium then this inability should be reflected in the kinetics of induction of allophanate hydrolase by these compounds. The kinetics of induction of allophanate hgdrolase by urea is shown in Fig. 4d ; a sharp increase in the rate of enzyme production follows a brief lag. When the analogues, formamide and hydantoic acid were used in place of urea (Fig. 4B), it was found that the lag was somewhat longer in the case of formamide and still longer in the case of hydantoic acid. Although the increased length of thr lag again suggests that the cells have difficulty in accumulating these compounds, it is clear that the addition of either analogue The inducers were added to the cultures at the indicated molar concent,rations, and one generation later the cells were harvested and the levels of allophanate hydrolase were determined. The inducers llsed were llrea (A), formamide (B), and hydant oic acid (C). resulted in a significant increase in the r&e of enzyme production.
n'hen examining the induction of enzymes by analogues, it is nc~essary to examine the metabolism of the analogues by the ~11s. From the structures of formamide and hydantoic acid, it is reasonable to expect that any degradation of these com-l~unds would lead to the production of ammonia.
Therefore, three cultures were grown on minimal medium containing urea, formamide, or hydantoic acid as the sole source of nitrogen. As shown in Fig. 5, urea was able to serve as the sole nitrogen source, but neither formamide nor hydantoic acid was able to sqq)ort growth when provided as the only nitrogenous coml)outtd. This indicates that neither of these compounds was drgraded to ammonia.
Although failure of the cells to utilize formamide suggests that it is not degraded by this yeast, the fact that the inducer of the urea degradation system is allophanate leads to the expectation that this urea analogue is carbosylated.
Consistent with this eqrctation, Whitney and Cooper (11)  formamide is able to substitute for urea in the urea-dependent cleavage of ATP. In addition, formyl urea and a&amide, the other urea analogues able to induce urea amido-lyase (Table  II), have been shown to support this ADP production in the absence of urea suggesting that' thrse compounds are also carboxylated.
Recently, it has been llossible to show l-his carbosylation directly (11). Thcsc data support the contention that the urea analogues callable of serving as inducers are carboxylated by urea carboxylase and suggest that the carboxylated products are rcsl>onsible for the induction of the urea degradation system.
If in fact this conclusion is correct then analogues of urea should not induce allophanate hydrolase in a urea carbosylasedefective strain.
This n-as tested by determining the level of allophanate hydrolase follon-ing incubation of a urea carboxylasedefective strain in the presence of urea and its analogues.
As shown in Table III  bosylase.
Since we demonstrated in Fig. 4 that there is a long lag before the onset of allophanate hydrolase induction when hydantoic acid is used as an inducer, the urea carboxylase-less mutant strain (M-62) was grown on minimal medium and the indicated compounds \I-vcre added at a sufficiently low cell density that when the cells were harvested the inducer had been present in the cult'ures for 2.5 to 3 generations instead of one generation as in the previous experiments.
As shown in Table IV under these conditions hydantoic acid was able to induce allophanate hydrolase approximately 2-fold in the mutant strain. Although the addition of urea and formamide resulted in a slight increase in enzyme level, it should be noted t'hat these levels are significantly below the level found in the culture which received hydantoic acid, whereas in all other experiments the levels found in cultures containing urea or formamide were severalfold higher than those found in cultures containing hydantoic acid. These data show that the urea analogue formamide must be carboxslated by urea carboxylase in order t,o serve as an inducer of allophanate while the allophanate analogue, hydantoic acid is able to serve as an inducer even in the absence of urea carbosylasc activit'y.
These data are thus consistent with the previous conclusion that the inducer of the multienzyme complex composed of urea carboxylase and allophanate hydrolase is the intermediate compound allophanatc.

Induction of Urea-degradative
Enqymes during Nitrogen Star-vationPUrea, as indicated above, is produced when S. cerevisiae is grow-11 v\-ith the amino acid arginille as a nitrogen source. Under thcsr growth conditions arginine is degraded to ornithine and urea by the enzyme arginase.
It was found, in our studies of the induction of arginase, that the starvation of the cells for ammonia resulted in an immediate dcrcpression of arginase.
It was further shown that this derepression was contingent upon the presence of an arginine pool in t'he cells; that is, when cells were starved of both arginine and ammonia, no immediate derepression of arginasc occurred, although the addition of the nonmetabolizable inducer homoarginine did result in enzyme production under these conditions.
The conclusion drawn from these data was that ammonia inhibits the induction of arginase and that under conditions of ammonia starvation, the pool of arginine normally present in cells grown on minimal medium is sufficient to cause induction of arginasc.
Thus the immediate derepression of enzyme following the onset of rlikogen starvation is in fact induction Since it was also observed that urea amido-lyase was dercpressed follovr ing t,he onset of nitrogen starvation, this phenomenon \Tas examined in further detail.
The derepression of allophanate hydrolase in wild type cells under conditions of nitrogen starvation is shown in Fig. 6R. Exponentially growing cells were filtered and one-half of the culture was resuspended in medium devoid of a nitrogen source; the other half of the culture was resuspended in the same medium but the nonmetabolizable inducer of the urea degradation system, formamidc was added.
The inclusion of a culture containing a nonmetabolizable inducer is a necessary control which indicates the level of enzyme synthesis possible in different strains under various conditions.
It can be seen that allophanate hydrolase becomes derepressed following the onset' of nitrogen starvation and that the inclusion of formamide in the starvatiorl medium stimulates the production of hydrolase. If t,his derepression occurs by the same mechanism as that of arginase, then the production of enzyme is due to induction by the urea or allophanate present in the cells. A major source of urea in cells undergoing nitrogen starvation is the large intracellular pool of arginine. The lag preceding the derepression of allophanate hydrolase in the wild type strain, Fig. 6$, might be due to the time necessary to synthesize some arginase and convert the arginine to urea and  I I I I I I I I I III  I I II  1   20  40  60  80  100  120  140  then to allophanate. It should be noted that this lag was much shorter when the inducer formamide was present in the starvation medium.
The production of urea from arginine can be eliminated by the use of a mutant strain lacking arginase activity. -4s can be seen in the experiment in Fig. BB, nitrogen starvation of such a mutant results in no immediate derepression of allophanate hydrolase.
In the control culture which received formamide, this enzyme could be induced indicating that the cells still have the capacity to synthesize this protein when provided with inducer.
Since it has been shown that the inducer of the urea-degradative system is in fact allophanat.e, it can be predicted that a mutant lacking urea carboxplase, and therefore, unable to produce allophanate from urea, would not be able to produce allophanate hydrolase under conditions of nitrogen starvation. Furthermore, the addition of formamide should not alter the level of enzyme found, since this compound, like urea, must be carboxylated before it is able to serve as an inducer.
These predictions are confirmed by the results depicted in Fig. 6C. When the mutant lacking urea carboxylase was deprived of ammonia, either in the presence or absence of formamide, there was no large increase in the amount of allophanate hydrolase found in the cells. The very slight increase seen is probably an increase in the basal level; this increase was observed in all of the experiments and is only visible in this case because the ordinate has been expanded. DISCUSSION We have shown that in strains containing a nonfunctional urea carboxylase, the synthesis of allophanate hydrolase cannot be induced by urea. In contrast, in wild type strains the level of this enzyme increases 8-to lo-fold upon addition of urea. The same response was observed with two nonmetabolizable analogues of urea. These data are consistent with the suggestion that the inducer of allophanate hydrolase is allophanate. In addition, urea carboxylase activity, in an allophanate hydrolasedefective strain, was observed to be at its fully induced level whether or not urea was added to the culture. This observation may be accounted for by suggesting that allophanate is also the inducer of urea carboxylase and that in the hydrolase-deficient organism allophanate, which cannot be degraded, accumulates as a result of the metabolism of arginine.
This conclusion can be questioned, however, if the reaction catalyzed by urea carboxylase is reversible.
Under this condition it would not be possible to distinguish whether urea or allophanate is the legitimate inducer.
While the possibility of urea being the inducer of urea carboxylase is considered unlikely (the carboxylation reaction is only slightly reversible) the only way to establish with certainty that allophanate is the inducer of urea carboxglase is to monitor the affect of adding urea, to wild type and carboxylase-defective strains, on the amount of immunochemically detectable urea carboxylase cross-reacting material.
The suggestion that allophanate acts as the inducer of both urea carboxylase and allophanate hydrolase is reasonable since preliminary evidence indicates that the structural genes responsible for the synthesis of these two enzymes are contiguous.3 l-he present situation is similar to that observed for induction of the first two enzymes responsible for tryptophan degradation in Pseudomonas (12) and induction of the histidine utilization enzymes in Aerobacter (13). Palleroni and Stanier (12) demonstrated that the inducer of the first two enzymes of tryptophan degradation was kynurenine, the product of the second cnzyrne in the pathway, and Schlesinger et al. (13) showed that the enzymes of the histidine-degradative pathway are induced by urocanic acid the product of the first enzyme in the pathway.
In these cases, however, the first activity of each pathway is irreversible, so that the conclusion is unambiguous.
In the previous paper, data were presented showing that the urea-degradative enzymes were apparently derepressed during growth of this organism under conditions of nitrogen starvation.
Here we have looked at this question more thoroughly and have 3 D. Evans, P. A. Whitney, and T. G. Cooper, unpublished observations. demonstrated that the apparent derepression of the urea-degradative system is contingent upon the presence of the inducer, allophanate.
During conditions of nitrogen starvation the intracellular pool of arginine is sufficient to effect the induction of arginase.
The production of arginase presumably allows the degradation of arginine to urea and subsequently to allophanate. Allophanate in turn brings about the induction of the ureadegradative system. The apparent derepression is, therefore, in fact an example of internal induction.