Vol. 137, No. 3
JOURNAL OF PACTERIOLOGY, Mar. 1979, p. 1447-1448 0021-9193/79/03-1447/02$02.00/0
Addition of Basic Amino Acids Prevents G-1 Arrest of Nitrogen-Starved Cultures of Saccharomyces cerevisiae TERRANCE G. COOPER,* CINDY BRITTON, LESLIE BRAND, AND ROBERTA SUMRADA Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received for publication 26 December 1978
Arginase-minus mutants of Saccharomyces cerevisiae were arrested in growth and accumulated at the unbudded G-1 stage of the cell cycle when starved for nitrogen. If, however, arginine was added to the culture medium at the time of starvation, growth ceased but the cells did not collect at the unbudded G-1 stage. We suggest that arginine addition prevented the cells from collecting at the G-1 stage by starving them for histidine and lysine, thereby inhibiting synthesis of proteins needed to complete the cell cycle. It has been hypothesized that yeast cells can monitor the levels of required nutrients in their environment to ensure their presence before initiating a new round of cell division (7). If the nutrient supply is insufficient, the cells remain at the beginning of the cell cycle (unbudded, G1 stage) until it increases. The response of wildtype and arginase-minus mutants of Saccharomyces cerevisiae to starvation for nitrogen or other required nutrients is cessation of growth and accumulation of most cells at the unbudded G-1 stage. This phenomenon has been termed G-1 arrest by Hartwell and his colleagues. Internally sequestered metabolites and turnover of preexisting macromolecules provide the nutrients needed to complete the cell cycle if starvation occurs after it has begun (6). Unger and Hartwell (7) developed an elegant means to search for the "signal" compound which acts directly or indirectly to control initiation of the cell cycle and to bring about G-l arrest. Their analysis centered on sulfate starvation which, like nitrogen starvation, results in G-1 arrest of cells. They reasoned that cells might sense a loss of external sulfate by detecting a decrease in an unidentified "signal" compound. By their rationale, blocking sulfate metabolism before synthesis of the "signal" compound would result in G-1 arrest due to its deficiency. Blocking sulfate metabolism at a point subsequent to synthesis of the "signal" compound would not result in G-1 arrest because the concentration of the "signal" compound would remain high and the cells would have no means of detecting the starvation. A similar rationale can be applied to G-1 arrest resulting from nitrogen deprivation. Deprivation of wild-type or urea carboxylase-deficient strains for nitrogen resulted in normal G-1 arrest (budded/unbudded ratio = 0.19). Addition of urea to
the urea carboxylase-deficient mutant at the time of deprivation did not alter the cell's ability to accumulate at the unbudded G-1 stage. However, when this experiment was repeated with arginine and an arginase-minus mutant, a quite different result was observed. G-1 arrest occurred normally on starving the mutant for nitrogen (Fig. 1). However, if arginine was added at the time of nitrogen deprivation, most cells stopped growing at randomly distributed points throughout the cell cycle; they did not accumulate in the G-1 stage of the cycle. Thus, addition of arginine to the medium prevented cells from completing the cell cycle and accumulating at G-1 even though it could not be metabolized. At face value, this is the result expected if arginine played an iinportant role in control of the cell cycle. Unfortunately, such an appealing conclusion is incorrect. When lysine and histidine were added along with arginine, G-1 arrest occurred almost normally (Fig. 1). Sumrada and Cooper (5) found that growth of S. cerevisiae on glucose-allantoin or proline media (nitrogen sources which cause only very mild nitrogen repression) was totally inhibited by the addition of a nondegradable basic amino acid to the culture medium. Inhibition of growth was not observed when highly repressive nitrogen sources such as asparagine or serine were used instead. Cells growing in glucose-proline medium and treated with lysine contained greatly reduced intracellular pools of histidine and arginine. Conversely, soluble lysine and histidine levels were severely reduced in arginase-deficient cells treated with arginine. When all three basic amino acids were added to the culture medium, growth and soluble amino acid pool sizes were normal. In view of these observations, we suggest that arginine addition to a nitrogen-starved, arginase447
1448
J. BACrERIOL.
NOTES
round of division. Basic amino acid inhibition of growth and cellular processes has been erroneously explained in several other ways (1-4), (I) and this has led to faulty hypotheses of metabolic regulation (4,8). Although we have not yet -i I established the precise molecular events by IJw WlJO which an excess of one basic amino acid de0 creases intracellular levels of the others, it is 1S aCM) that the ability of arginine to prevent probable arginase-minus cells from accumulating at G-1 aOWa D is a manifestation of this phenomenon rather m than a reflection of some unique role it might a) z play in control of the cell cycle. The starvation D3 conditions described here likely represent a more severe but analogous case of the ornithine o inhibition of growth we observed with cells growing in glucose-proline medium (5). In that situation, protein synthesis was depressed 75% (5). The purpose of this report has been to correlate our observations of S. cerevisiae physiology with seemingly unrelated experiments concern6 4 8 2 control of the cell division cycle. The indirect HOURS OF STARVATION ing effects of arginine on the cell cycle could be FIG. 1. Effect of basic amino acids on G-1 arrest easily, but mistakenly, interpreted as being perof arginase-minus strains of S. cerevisiae starved for tinent to an understanding of the molecular ammonia. Two cultures of a diploid arginase-minus events controlling initiation of cell division in strain (M58) were grown in minimal, glucose-ammo- the absence of this correlation. nia medium (5) containing a reduced concentration of ammonium sulfate (0.05%). This concentration of ammonium sulfate was selected because it permits rapid onset ofnitrogen starvation without increasing the doubling time of the unstarved culture. One of the two cultures also contained the basic amino acids lysine and histidine each at a final concentration of 200 mg/liter. At a cell density of 25 Klett units (ca. 1 x 167 cells/ml), the cells from both cultures were harvested by filtration, washed three times with 50 ml of prewarmed, preaerated medium devoid of ammonid and resuspended in fresh ammonia-free medium. The culture that was pregrown in the presence of lysine and histidine was also starved in the presence of these amino acids. Once the cells had been resuspended, the culture that was suspended in medium devoid of amino acids was split into two portions. One portion received no further additions (U), and arginine (200 mg/l) was added to the other portion (). An identical concentration of arginine was also added to the culture that contained the basic amino acids (0). Thereafter, 2-ml portions of each of the three cultures were removed at the times indicated in the figure and added to an equal volume of a solution containing 0.15 MNaCI and 3.7% formaldehyde. Subsequent to this point, the distribution of budded and unbudded cells was determined as described by Sumrada and Cooper (5). minus culture prevented the cells from accumulating in G-1 by starving them for histidine and lysine, thereby inhibiting synthesis of proteins needed to complete the cell cycle or to act as "signal" compounds needed to initiate a new
L. H. Hartwell and members of his group have independently observed the arginine-mediated inhibition of G-1 arrest of nitrogen-starved, arginase-deficient cells. This work was supported by Public Health Service grants GM-19386 and GM-20693 from the National Institute of General Medical Sciences. T.G.C. was the recipient of a Research Career De-
velopment award K04-GM-00091 also from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Bourgeois, C. 1969. Influence de la lysine sur la croissance ce Saccharomyces cerevisiae. Bull. Soc. Chim. Biol. 61:935-949. 2. Bourgeois, C. M., and D. R. Thouvenot. 1970. Effets de la lysine sur la synthese et l'activite de l'arginase et de l'omithone trnsaminase chez Saccharomyces cerevisiae. Eur. J. Biochem. 15:140-145. 3. Hunter, A., and C. E. Downs. 1945. The inhibition of arginase by amino acids. J. Biol. Chem. 157:427-446. 4. Ramos, R., P. Thuriax, J. M. Wiame, and J. Bechet. 1970. The participation of ornithine and citrulline in the regulation of arginine metabolism in Saccharomyces cerevisiae. Eur. J. Biochem. 12:40 47. 5. Sumrada, R., and T. G. Cooper. 1978. Basic amino acid inhibition of cell division and macromolecular synthesis in Saccharomyces cerevisiae. J. Gen. Microbiol. 108:
45-56. 6. Sumrada, ., and T. G. Cooper. 1978. Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae. J. Bacteriol.
136:234-246. 7. Unger, M. W., and L H. Hartwell. 1976. Control of cell division in Saccharomyces cerevisiae by methionyltRNA. Proc. Natl. Acad. Sci. U.S.A. 73:1664-1668. 8. Vaca, G., and J. Mora. 1977. Nitrogen regulation of arginase in Neurospora crassa. J. Bacteriol. 131:719725.
JOURNAL OF PACTERIOLOGY, Mar. 1979, p. 1447-1448 0021-9193/79/03-1447/02$02.00/0
Addition of Basic Amino Acids Prevents G-1 Arrest of Nitrogen-Starved Cultures of Saccharomyces cerevisiae TERRANCE G. COOPER,* CINDY BRITTON, LESLIE BRAND, AND ROBERTA SUMRADA Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received for publication 26 December 1978
Arginase-minus mutants of Saccharomyces cerevisiae were arrested in growth and accumulated at the unbudded G-1 stage of the cell cycle when starved for nitrogen. If, however, arginine was added to the culture medium at the time of starvation, growth ceased but the cells did not collect at the unbudded G-1 stage. We suggest that arginine addition prevented the cells from collecting at the G-1 stage by starving them for histidine and lysine, thereby inhibiting synthesis of proteins needed to complete the cell cycle. It has been hypothesized that yeast cells can monitor the levels of required nutrients in their environment to ensure their presence before initiating a new round of cell division (7). If the nutrient supply is insufficient, the cells remain at the beginning of the cell cycle (unbudded, G1 stage) until it increases. The response of wildtype and arginase-minus mutants of Saccharomyces cerevisiae to starvation for nitrogen or other required nutrients is cessation of growth and accumulation of most cells at the unbudded G-1 stage. This phenomenon has been termed G-1 arrest by Hartwell and his colleagues. Internally sequestered metabolites and turnover of preexisting macromolecules provide the nutrients needed to complete the cell cycle if starvation occurs after it has begun (6). Unger and Hartwell (7) developed an elegant means to search for the "signal" compound which acts directly or indirectly to control initiation of the cell cycle and to bring about G-l arrest. Their analysis centered on sulfate starvation which, like nitrogen starvation, results in G-1 arrest of cells. They reasoned that cells might sense a loss of external sulfate by detecting a decrease in an unidentified "signal" compound. By their rationale, blocking sulfate metabolism before synthesis of the "signal" compound would result in G-1 arrest due to its deficiency. Blocking sulfate metabolism at a point subsequent to synthesis of the "signal" compound would not result in G-1 arrest because the concentration of the "signal" compound would remain high and the cells would have no means of detecting the starvation. A similar rationale can be applied to G-1 arrest resulting from nitrogen deprivation. Deprivation of wild-type or urea carboxylase-deficient strains for nitrogen resulted in normal G-1 arrest (budded/unbudded ratio = 0.19). Addition of urea to
the urea carboxylase-deficient mutant at the time of deprivation did not alter the cell's ability to accumulate at the unbudded G-1 stage. However, when this experiment was repeated with arginine and an arginase-minus mutant, a quite different result was observed. G-1 arrest occurred normally on starving the mutant for nitrogen (Fig. 1). However, if arginine was added at the time of nitrogen deprivation, most cells stopped growing at randomly distributed points throughout the cell cycle; they did not accumulate in the G-1 stage of the cycle. Thus, addition of arginine to the medium prevented cells from completing the cell cycle and accumulating at G-1 even though it could not be metabolized. At face value, this is the result expected if arginine played an iinportant role in control of the cell cycle. Unfortunately, such an appealing conclusion is incorrect. When lysine and histidine were added along with arginine, G-1 arrest occurred almost normally (Fig. 1). Sumrada and Cooper (5) found that growth of S. cerevisiae on glucose-allantoin or proline media (nitrogen sources which cause only very mild nitrogen repression) was totally inhibited by the addition of a nondegradable basic amino acid to the culture medium. Inhibition of growth was not observed when highly repressive nitrogen sources such as asparagine or serine were used instead. Cells growing in glucose-proline medium and treated with lysine contained greatly reduced intracellular pools of histidine and arginine. Conversely, soluble lysine and histidine levels were severely reduced in arginase-deficient cells treated with arginine. When all three basic amino acids were added to the culture medium, growth and soluble amino acid pool sizes were normal. In view of these observations, we suggest that arginine addition to a nitrogen-starved, arginase447
1448
J. BACrERIOL.
NOTES
round of division. Basic amino acid inhibition of growth and cellular processes has been erroneously explained in several other ways (1-4), (I) and this has led to faulty hypotheses of metabolic regulation (4,8). Although we have not yet -i I established the precise molecular events by IJw WlJO which an excess of one basic amino acid de0 creases intracellular levels of the others, it is 1S aCM) that the ability of arginine to prevent probable arginase-minus cells from accumulating at G-1 aOWa D is a manifestation of this phenomenon rather m than a reflection of some unique role it might a) z play in control of the cell cycle. The starvation D3 conditions described here likely represent a more severe but analogous case of the ornithine o inhibition of growth we observed with cells growing in glucose-proline medium (5). In that situation, protein synthesis was depressed 75% (5). The purpose of this report has been to correlate our observations of S. cerevisiae physiology with seemingly unrelated experiments concern6 4 8 2 control of the cell division cycle. The indirect HOURS OF STARVATION ing effects of arginine on the cell cycle could be FIG. 1. Effect of basic amino acids on G-1 arrest easily, but mistakenly, interpreted as being perof arginase-minus strains of S. cerevisiae starved for tinent to an understanding of the molecular ammonia. Two cultures of a diploid arginase-minus events controlling initiation of cell division in strain (M58) were grown in minimal, glucose-ammo- the absence of this correlation. nia medium (5) containing a reduced concentration of ammonium sulfate (0.05%). This concentration of ammonium sulfate was selected because it permits rapid onset ofnitrogen starvation without increasing the doubling time of the unstarved culture. One of the two cultures also contained the basic amino acids lysine and histidine each at a final concentration of 200 mg/liter. At a cell density of 25 Klett units (ca. 1 x 167 cells/ml), the cells from both cultures were harvested by filtration, washed three times with 50 ml of prewarmed, preaerated medium devoid of ammonid and resuspended in fresh ammonia-free medium. The culture that was pregrown in the presence of lysine and histidine was also starved in the presence of these amino acids. Once the cells had been resuspended, the culture that was suspended in medium devoid of amino acids was split into two portions. One portion received no further additions (U), and arginine (200 mg/l) was added to the other portion (). An identical concentration of arginine was also added to the culture that contained the basic amino acids (0). Thereafter, 2-ml portions of each of the three cultures were removed at the times indicated in the figure and added to an equal volume of a solution containing 0.15 MNaCI and 3.7% formaldehyde. Subsequent to this point, the distribution of budded and unbudded cells was determined as described by Sumrada and Cooper (5). minus culture prevented the cells from accumulating in G-1 by starving them for histidine and lysine, thereby inhibiting synthesis of proteins needed to complete the cell cycle or to act as "signal" compounds needed to initiate a new
L. H. Hartwell and members of his group have independently observed the arginine-mediated inhibition of G-1 arrest of nitrogen-starved, arginase-deficient cells. This work was supported by Public Health Service grants GM-19386 and GM-20693 from the National Institute of General Medical Sciences. T.G.C. was the recipient of a Research Career De-
velopment award K04-GM-00091 also from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Bourgeois, C. 1969. Influence de la lysine sur la croissance ce Saccharomyces cerevisiae. Bull. Soc. Chim. Biol. 61:935-949. 2. Bourgeois, C. M., and D. R. Thouvenot. 1970. Effets de la lysine sur la synthese et l'activite de l'arginase et de l'omithone trnsaminase chez Saccharomyces cerevisiae. Eur. J. Biochem. 15:140-145. 3. Hunter, A., and C. E. Downs. 1945. The inhibition of arginase by amino acids. J. Biol. Chem. 157:427-446. 4. Ramos, R., P. Thuriax, J. M. Wiame, and J. Bechet. 1970. The participation of ornithine and citrulline in the regulation of arginine metabolism in Saccharomyces cerevisiae. Eur. J. Biochem. 12:40 47. 5. Sumrada, R., and T. G. Cooper. 1978. Basic amino acid inhibition of cell division and macromolecular synthesis in Saccharomyces cerevisiae. J. Gen. Microbiol. 108:
45-56. 6. Sumrada, ., and T. G. Cooper. 1978. Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae. J. Bacteriol.
136:234-246. 7. Unger, M. W., and L H. Hartwell. 1976. Control of cell division in Saccharomyces cerevisiae by methionyltRNA. Proc. Natl. Acad. Sci. U.S.A. 73:1664-1668. 8. Vaca, G., and J. Mora. 1977. Nitrogen regulation of arginase in Neurospora crassa. J. Bacteriol. 131:719725.
