Control of arginine metabolism in Neurospora. Induction of ornithine aminotransferase.

In Neurosporu, most of the intracellular arginine pool is sequestered in osmotically sensitive organelles, the “vesicles.” In this paper, we describe the factors influencing the induction of the arginine degradative enzyme ornithineoxo-acid aminotransferase (EC 2.6.1.13). Upon addition of arginine to the growth medium, increased activity for the enzyme appears after a 30to 40-min lag. The initial rate of enzyme accumulation remains constant for approximately 80 min despite a continuously increasing intracellular arginine pool. Approximately 30 min following complete expansion of the arginine pool, the rate of enzyme accumulation increases abruptly until the steady state-induced level is achieved. Induction requires both protein and RNA synthesis. Synthesis of ornithine aminotransferase-forming capacity commences within minutes following addition of arginine to the growth medium. The initial rate of accumulation of this forming capacity is related to the rate of arginine uptake from the growth medium. Induced levels of the enzyme begin to decline 30 min after the removal of arginine from the growth medium. The rate of accumulation of ornithine aminotransferase appears to revert to the basal level within minutes following arginine removal from the growth medium. Reversion to the basal rate of accumulation occurs while the intracellular arginine concentration is still higher than that required for enzyme induction. We suggest that compartmentation of arginine plays a significant role in controlling arginine metabolism in Neurospora.

In Neurosporu, most of the intracellular arginine pool is sequestered in osmotically sensitive organelles, the "vesicles." In this paper, we describe the factors influencing the induction of the arginine degradative enzyme ornithineoxo-acid aminotransferase (EC 2.6.1.13). Upon addition of arginine to the growth medium, increased activity for the enzyme appears after a 30-to 40-min lag. The initial rate of enzyme accumulation remains constant for approximately 80 min despite a continuously increasing intracellular arginine pool. Approximately 30 min following complete expansion of the arginine pool, the rate of enzyme accumulation increases abruptly until the steady state-induced level is achieved. Induction requires both protein and RNA synthesis. Synthesis of ornithine aminotransferase-forming capacity commences within minutes following addition of arginine to the growth medium. The initial rate of accumulation of this forming capacity is related to the rate of arginine uptake from the growth medium.
Induced levels of the enzyme begin to decline 30 min after the removal of arginine from the growth medium. The rate of accumulation of ornithine aminotransferase appears to revert to the basal level within minutes following arginine removal from the growth medium. Reversion to the basal rate of accumulation occurs while the intracellular arginine concentration is still higher than that required for enzyme induction.
We suggest that compartmentation of arginine plays a significant role in controlling arginine metabolism in Neurospora.
Many prokaryotic organisms differ from typical eukaryotic cells in having much smaller pools of metabolites, especially amino acids. These levels are high enough to satisfy the concentration requirements of the enzymes which use the amino acids for protein synthesis. However, little catabolism occurs because the catabolic enzymes are neither induced nor very active at such low concentrations.
One can hypothesize that small intracellular pools provide prokaryotes with great * This work was supported in part by Grant GB43240 from the National Science Foundation and bv Grant 3072 from the Committee on Research, Academic Senate, Umversity of California, Los Angeles. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sensitivity to changes in the composition of the growth medium. For example, amino acid concentrations in Escherichia coli are typically on the order of 0.05 to 1 mM (1). If E. coli growing in minimal medium is given exogenous arginine. uptake of only a small amount of the amino acid from the medium should be sufficient to cause an increase in the concentration of the intracellular pool large enough to initiate a regulatory response at the gene level. Results consistent with this hypothesis were obtained by Krzyzek and Rogers (2) in their study of the repression of the arginine biosynthetic enzymes by arginine in E. coli. Their results indicated that the rate of messenger RNA synthesis for the arginine biosynthetic enzymes fell to the repressed level immediately after the addition of arginine to the cell culture. Thus, as was predicted, uptake of only a small amount of arginine was sufficient to begin the regulatory response.
In eukaryotes, large storage pools of metabolic intermediates are often present. In Neurosporu, the intracellular concentration of arginine would be 8 mM in cell water if it were equally distributed throughout the cell (3). This concentration is large enough to maximally inhibit arginine biosynthesis (41, yet biosynthesis occurs. Furthermore, the amino acids coexist with large amounts of arginase (EC 3.5.3.1) and ornithine aminotransferase, the first two enzymes of arginine degradation, yet little catabolism occurs (3, 5). These apparently contradictory aspects of arginine metabolism in Neurospora have been investigated in depth in recent years. The study has produced detailed information on the interrelationship between subcellular structure and arginine metabolism. The major results are conveniently summarized in Fig. 1. Most of the large arginine pool is located inside a membrane-bound organelle which has been termed "the vesicle" (6). The final step of arginine biosynthesis occurs in the cytosol (7). Most of the biosynthetically generated arginine is incorporated into protein, and the remainder is taken up by the vesicle. The biosynthetic enzyme subject to feedback inhibition by arginine, the arginine catabolic enzymes, and the arginine charging enzyme are cytosolic (7,8). Protein synthesis occurs in the absence of significant catabolism because the concentration of cytosolic arginine is maintained at a level too low for binding to arginase but sufficient for binding to the charging enzyme (9, 10). Catabolism occurs only when arginine is present in the growth medium (3,5 and catabolism occurs (3, 12). When Neurospora is grown in minimal medium, the confinement of most of the intracellular arginine to the vesicle leaves the concentration of the cytosolic arginine pool quite low and on the order of the concentration of amino acid pools in bacteria (9). When Neurospora is grown in arginine-supplemented medium, the cytosolic arginine concentration is high (11). Arginine from the growth medium directly enters the cytosol (9). Neurospora should be able to respond quickly and efficiently to the availability of arginine if its responses were directed by the concentration of the cytosolic arginine pool. Catabolism of arginine has been shown to fulfill these expectations (12).
The experiments reported here were designed to determine how rapidly enzyme activities change in response to alterations in the availability of arginine. The response was quantitated by following the activity of ornithine aminotransferase or the forming capacity for the enzyme after addition or removal of arginine from the medium. The results are consistent with enzyme activity being controlled by the cytosolic arginine concentration.
The latter appears to be related to the rate of arginine uptake from the growth medium.

Arginine
Uptake and Induction of Ornithine Aminotransferase- Fig. 2 shows the increase in activity of ornithine aminotransferase and the intracellular arginine pool at various times after the addition of arginine to the growth medium. Enzyme activity increased linearly for approximately 90 min following an initial lag of 30 to 40 min. Similar results have been observed for arginase (12  To further characterize the induction, the effect of inhibitors of RNA and protein synthesis were investigated. Table I shows that 6-methylpurine, an inhibitor of RNA synthesis (19), and cycloheximide, an inhibitor of protein synthesis (20), prevented a significant rise in ornithine aminotransferase specific activity. Thus it appears that induction of the enzyme requires both RNA and protein synthesis.
These results demonstrate that the appearance of increased enzyme activity occurs only after a sizeable increase in the intracellular arginine pool. Induction of ornithine aminotransferase might not begin until the arginine pool has reached a sufficiently high intracellular concentration. Once the pool reached this level, processes involved in the increased rate of enzyme accumulation could proceed rapidly. However, it is also conceivable that ornithine aminotransferase induction might begin soon after arginine addition and long before major expansion of the intracellular arginine pool. The 30-to 40-min lag preceding the rise in enzyme specific activity could simply be a reflection of the time required for expression of increased enzyme-forming capacity as accumulated enzyme. The experiments below were designed to distinguish between these alternatives.

Kinetics
of Znduction of Ornithine Aminotransferase mRNA -In order to determine the time of induction initiation, an attempt was made to measure the level of ornithine aminotransferase-forming capacity at intervals following arginine supplementation.
This required an assay for specific enzyme forming capacity. In the experiments reported here, enzyme-forming capacity was assayed by its ability to yield enzyme. Kepes (21) and Cybis and Weglenski (22) have successfully used this method. In order to use an assay of this type, it was necessary to determine the time required to get maximum expression of enzyme-forming capacity as its enzyme product. Cells growing in minimal medium were exposed to arginine for 80 min, washed, and transferred to argininefree medium. The specific activity of ornithine aminotransferase was examined at various intervals after the transfer (Fig. 3). The specific activity rose, reached a peak approximately 90 min after the transfer, and subsequently fell. This behavior is independent of the time of exposure to arginine. These results indicate that full expression of enzyme-forming capacity occurs after 90 min of incubation in arginine-free medium.
Enzyme activity present 90 min after transfer was used as a measure of the amount of ornithine aminotransferase-speciflc enzyme-forming capacity produced during and after exposure of cells growing in minimal medium to arginine for various periods of time. The activity has been plotted uersus the time of exposure to arginine (Fig. 4). The results are compared with the normal induction kinetics. Increased enzyme-forming capacity for ornithine aminotransferase appears to be present immediately after arginine addition. Thus, the induction must begin after uptake of only a small amount of arginine (see Fig. 2). In addition, the accumulation of enzymeforming capacity was constant for at least 60 min and was not affected by the continuous increase in the size of the arginine pool.
Effect of Inducer Removal on Ornithine Aminotransferase Activity-Following the study of the response to arginine addition, response to arginine removal was investigated.
The goal of the investigation was to determine the relation between the size of the intracellular arginine pool and the level of ornithine aminotransferase. This information was obtained by following the enzyme activity and the fall of the arginine pool when wild type cells grown in arginine-supplemented medium were transferred to arginine-free medium. Fig. 5 Control o'"L-----l 0 IO  shows that the size of the arginine pool began to decrease immediately after the transfer and fell toward the basal level (20 nmol/mg dry weight). The activity of ornithine aminotransferase began to drop 30 min after the transfer and also decreased toward the basal level (0.2 pmol/min/mg of protein). The activity of the enzyme fell with a half-life of 3.4 h, which is longer than the 2.05-h doubling time of the cells. This longer half-life suggested that the decay in specific activity was not due to rapid enzyme turnover. To further substantiate this conclusion, the effect of cycloheximide on the decline in enzyme activity was investigated.  specific activity of ornithine aminotransferase. Thus, it appears that the induced enzyme is quite stable. These results suggest that the decline in enzyme activity is not due to rapid degradation or enzyme turnover, but probably results from a reduction in the rate of active enzyme production. This would cause the specific activity of the enzyme to drop as a result of dilution by newly synthesized protein.
Evidence supporting this mode of decay comes from the close agreement of the experimental results with those predicted by the following model. The decline in ornithine aminotransferase activity is the result of a decrease in the rate of its production and resulting dilution by new protein; the rate of production decreases in the absence of inducer from the induced to the basal rate within minutes after the transfer to arginine-free medium; previously produced enzyme-forming capacity is expressed and then decays; ornithine aminotransferase is infinitely stable. It is possible to calculate theoretical curves based on these assumptions. If one assumes a negligible half-life for enzyme-forming capacity, enzyme specific activity at any time following inducer removal should conform where SA is the specific enzyme activity, k is the growth constant calculated for cells with a generation time of 123 min, and t is the time after transfer. If one assumes that expression of pre-existing enzyme forming capacity requires 30 min (Fig. 2), the equation becomes: sA _ SA (induced) x e ?W + SA (uninduced) x (eli' ~ e'"'") I 81 The two curves differ only in the length of time following the transfer to arginine-free medium for the expression of preexisting enzyme-forming capacity. In one curve, the assumption is made that this occurs immediately after the transfer and in the other, 30 min after the transfer. The theoretical curves and the experimental results are shown in Fig. 6. Since most of the experimental points fall on or between the two curves, it appears that the rate of ornithine aminotransferase production decreases to the basal level within minutes after the transfer to arginine-free medium. Fig. 5 shows that the size of the arginine pool 30 min after transfer to argininefree medium is about 100 nmol/mg dry weight, more than 5 times the size of the pool in cells grown in minimal medium.
Rate of Arginine Uptake and Enzyme Induction-The results described above suggest that production of ornithine aminotransferase is not proportional to the size of the intracellular arginine pool. To explore this apparent contradiction, we have examined the initial rate of enzyme accumulation under conditions in which the rate of arginine uptake has been varied. In the bat mutant, arginine is taken up from the medium only by the general amino acid permease (13). The arginine uptake rate can be controlled by using other amino  8 (right). Induction of ornithine aminotransferase and rate of arginine uptake. The bat mutant growing in minimal medium was exposed to 1.0 mM arginine and various concentrations of glycine for 60 min. The cells were collected, washed, transferred to minimal medium for 90 min, and then assayed for ornithine aminotransferase activity.
acids to compete with arginine for uptake by this transport system. Cells growing in minimal medium were simultaneously exposed to arginine and various concentrations of glytine for a short period of time. Fig. 7 shows that arginine uptake rate during the exposure period was proportional to the molar fraction of arginine in the medium.
In a similar experiment, the rate of ornithine aminotransferase accumulation was determined by assaying for the total enzyme-forming capacity produced in a given length of time. Cells growing in minimal medium were exposed to arginine and various concentrations of glycine for 60 min, then washed and transferred to minimal medium for 90 min to allow full expression of the enzyme-forming capacity. Fig. 8 shows that the rate of accumulation was determined by the molar fraction of arginine in the medium. The previous experiment indicated that the rate of arginine uptake was also determined by the molar fraction of arginine. Therefore, it follows that the rate of ornithine aminotransferase accumulation is determined by the rate of arginine uptake into the cell.

DISCUSSION
The results in Fig 2 indicate that the rate of ornithine aminotransferase accumulation does not increase for 30 to 40 min following the addition of its inducer, arginine, to the growth medium. This is in contrast to the 5-to lo-min lag observed for kynureninase (EC 3.7.1.3) in Neurospora (23) and the 3-to 4-min lag for arginase and allophanate hydrolase in Saccharomyces cereuisiae (24-27). These lags may represent the time required for transcription, processing, and translation of specific mRNA molecules. This hypothesis remains to be investigated. In each case, however, the increased rate of enzyme accumulation appears to be constant for an extended period (when corrected for cell growth) after the lag phase. In the case of ornithine aminotransferase, this initial Control of Arginine Metabolism in Neurospora 6979 constant induced rate of accumulation occurs despite a continually increasing intracellular arginine pool (Fig. 2). Approximately 30 min after maximal expansion of the arginine pool, the rate of enzyme accumulation increases abruptly and continues at this increased rate until the steady state level characteristic of cells growing in arginine-supplemented medium is achieved. These results suggest that the concentration of arginine (or its derivative) which controls the rate of enzyme production remains constant during the steady state induction phase but increases significantly when the intracellular pool reaches its maximal value. This is reflected following a 30-min lag by an increase in the rate of enzyme accumulation.
The long lag phase observed (Fig. 2) might reflect the establishment of an inducing level of intracellular arginine or the time required for expression of enzyme-forming capacity as active enzyme. Fig. 3 shows that 90 min are required for complete expression following the removal of the inducer, arginine. This compares to the 16 min observed for kynureninase (23) and 10 min for allophanate hydrolase (26). In all cases (22)(23)(24)26; also see below) it appears that the increased rate of production of enzyme-forming capacity ceases abruptly following inducer removal. Using these results, it has been possible to examine the initiation of the increase in the rate of production of ornithine aminotransferase-forming capacity (Fig. 4). It appears that the increased rate of ornithine aminotransferase-forming capacity accumulation is initiated within minutes following arginine supplementation, remains constant, and parallels enzyme accumulation for at least 60 min. This occurs despite a steadily increasing intracellular arginine concentration.
It appears unlikely that this response is mediated by the total intracellular arginine concentration. When arginine is removed from arginine-supplemented medium, ornithine aminotransferase activity begins to decline following a lag of approximately 30 min (Fig. 5). This occurs at a time when the intracellular arginine pool is approximately 5 times that found in cells growing in minimal medium. Fig. 6 compares the observed decay of ornithine aminotransferase specific activity with models based upon an abrupt shift to the noninduced rate of enzyme production. In view of the long time required for expression of ornithine aminotransferase-forming capacity (Fig. 31, it is likely that much of this lag period reflects expression of such capacity formed in the presence of arginine. It would appear that the rate of production of ornithine aminotransferase shifts to the noninduced rate shortly after arginine is removed from the growth medium and long before the intracellular pool level drops below that required for induction (Figs. 2 and 4).
These results suggest that ornithine aminotransferase production is independent of the total intracellular arginine concentration, but instead depends on the presence or absence of arginine uptake from the medium. It has previously been shown that much of the intracellular arginine pool of Neurosporu is sequestered within a membrane-enclosed organelle (6). A schematic diagram of the structural and locational relationships between the relevant enzymes and arginine is shown in Fig. 9. During growth in minimal medium, the cytosolic pool remains small, catabolic enzymes are at a basal level, and catabolism is almost nonexistent (3,5,9). During growth in arginine-supplemented medium, the cytosolic arginine concentration increases much more than that of the total arginine pool, enzymes are induced to a small degree (4fold), and catabolism proceeds rapidly (3,11,12). During transitions between growth in the presence and absence of arginine, the rate of catabolism responds fully within minutes (12). The model shown in Fig. 9 suggests that control of the level of gene expression might also respond to changes in the cytosolic arginine concentration. During growth in minimal medium, the cytosolic arginine concentration would remain low and ornithine aminotransferase would be uninduced.
Upon arginine addition to the growth medium, uptake would occur, the cytosolic arginine concentration would rise until it reached a steady state value dependent on the rates of arginine entrance into and exit from the cytosol. This steady state value is likely to largely depend on the rate of uptake from the medium. The immediate response to arginine addition shown by ornithine aminotransferase-forming capacity and the constant increase during pool expansion are consistent with this model (Fig. 4). Once the "vesicular pool" becomes fully occupied, the cytosolic arginine concentration must rise further since one means of its exit is now eliminated. This is reflected in an increased rate of production of enzyme-forming capacity. The latter results in new enzyme following the 30-min lag required for its expression (Fig. 2). The increased cytosolic arginine concentration will control the rate of uptake by transinhibition until the steady state level characteristic of long term growth in arginine-supplemented medium is achieved. If arginine is removed from the growth medium, the cytosolic concentration will drop as uptake ceases. This results in rapid cessation of catabolism (12) and return to the basal rate of ornithine aminotransferase production (Fig. 6). A basic tenet of this model is that responses governed by the cytosolic arginine concentration will be affected by the rate of arginine uptake from the medium. The rate of arginine uptake can be controlled by competitive inhibitors of its Control of A&nine Metabolism in Neurospora transport system. This is best accomplished using a mutant which used a nonspecific transport system for arginine. Such a mutant is the bat strain which can transport arginine only via the general amino acid permease (13). The feasibility of this approach has been demonstrated (Fig. 7). The relationship between the initial rate of accumulation of enzymeforming capacity and the arginine uptake rate clearly suggests that the rate of uptake affects the cytosolic arginine concentration and the induction process which responds to this concentration (Fig. 8).
It remains to be examined why the response of arginase (12) and ornithine aminotransferase is significantly slower than that of kynureninase in Neurospora and arginase and allophanate hydrolase in yeast. Mechanistic possibilities include: slower rates of mRNA processing, activation or assembly of completed enzyme molecules, or slow insertion of required cofactors. These possibilities remain to be investigated. From a functional point of view, two features distinguish the kynureninase catabolic system from the arginine degradative pathway. First, the level of the enzyme in cells growing in minimal medium is low, and the activity increases 120-fold in the presence of inducers (23). Second, the intracellular tryptophan and kynurenine pools are very small during growth in minimal medium (23). These observations suggest that addition of tryptophan to the growth medium will quickly affect the total intracellular tryptophan concentration, but that efficient metabolism will require significant enzyme induction. In contrast, arginase, ornithine aminotransferase, and arginine levels are high in cells growing in minimal medium. The enzymes are only induced 4-fold and the total arginine pool expands only 7-fold (3, 11). Compartmentation ( Fig. 9) coupled with pre-existing enzyme allows Neurospora to respond quickly to changes in the availability of arginine (12). Although there is some evidence for the compartmentation of tryptophan in Neurospora, its significance is not clear (28).
In yeast, arginine is compartmentalized in the vacuole, an organelle similar to the vesicle of Neurospora (29). The difference in response would appear to be a consequence of the necessity for enzyme induction for maximal catabolism since enzyme levels are low during growth in minimal medium (30). Compartment&ion would appear to add an additional means of controlling amino acid metabolism. In arginine metabolism in Neurospora, it would appear to play a decisive role, whereas in yeast it may contribute significantly although enzyme induction appears to play a major role. The rationale for such differences may reside in the life-styles of the organisms. Neurospora's arginine metabolism appears to be ideally suited to fluctuating environments in which arginine alternately becomes available or is depleted. The overall significance of compartmental processes in higher eukaryotes remains to be investigated.