Vol. 136, No. 1
JOURNAL OF BACTERIOLOGY, Oct. 1978, p. 234-246 0021-9193/78/0136-0234$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Control of Vacuole Permeability and Protein Degradation by the Cell Cycle Arrest Signal in Saccharomyces cerevisiae ROBERTA SUMRADA AND TERRANCE G. COOPER* Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received for publication 28 April 1978
Saccharomyces cerevisiae responds to deprivation of nutrients by arresting cell division at the unbudded Gl stage. Cells situated outside of Gl at the time of deprivation 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 afactor 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 a cells or a/a diploid strains were treated with a-factor. These data suggest that release of vacuolar constituents and protein turnover may be regulated by the Gl arrest signal. Many "higher" and "lower" eucaryotic organisms control their cell division cycle in response to the availability of organic and inorganic nutrients. Saccharomyces cerevisiae respond to starvation for glucose, ammonia, sulfate, phosphate, biotin, or potassium by arresting cell division at the unbudded Gl stage (20, 49). Johnston et al. observed that nutrient-deprived cells situated outside of Gl were able to complete their cell cycles (21). However, they neither initiated new division cycles nor completed any of the three known gene-controlled steps (cdc28, cdc4, or cdc7 gene product function) suggested by Reid and Hartwell to be among the earliest detectable events in division (30). It has been hypothesized that yeast celLs have some mechanism for monitoring the levels of required nutrients to ensure their presence before a new round of cell division is initiated (20). In an effort to identify the biochemical events asociated with monitoring sulfate sufficiency and the decision to "start" a round of division, Unger and Hartwell used mutants blocked in sulfate reduction, methionine biosynthesis, and methionyl-tRNA acylation (39). They concluded that the pivotal regulatory process occurred subsequent to the charging of tRNA. They could not proceed further to determine whether or not protein synthesis itself was involved in the de234
cision-making process of the cells, because loss of protein synthesis results in immediate cessation of growth and division irrespective of the position of the cells in the division cycle. Since their approach required that cells be able to complete division once they had successfuly passed the regulatory point, further progress with this experimental format was not possible. Although nutrient supply plays an important role in control of the cell cycle, it is by no means exclusive. S. cerevisiae have been shown to mate only when both mating cells are situated in Gl (10), which means that asynchronously growing a and a cells must synchronize their cell cycles before they can mate. This problem has apparently been solved by the elaboration of pheromones, which transiently arrest growth at Gl. Cells of the a mating type constitutively secrete a tridecapeptide hormone, a-factor, into the medium. Cells of the a mating type that come in contact with a-factor complete their cell division cycle and arrest as unbudded Gl cells. Arrest lasts for a period of time which is dependent, among other things, on the concentration of afactor encountered (12, 49). An analogous set of reciprocal events bring about Gl arrest of a celLs, although they have not yet been as well-studied biochemically (5, 12). The important feature of nutrient starvation
VOL. 136, 1978
CONTROL BY THE CELL CYCLE ARREST SIGNAL
and treatment with mating pheromones is that growth is arrested at the same point in both cases. This raises the possibility that some of the sensing and control elements may be shared in common, even though the two processes causing arrest are quite different. Our initial interest in the problem of G1 arrest was motivated by the question: How could cells complete division in the absence of sufficient external nutrients? We considered two possible nutrient sources that could be tapped during times of external deprivation: the cell vacuole and turnover of previously synthesized constituents. Major reserves of S-adenosylmethionine, arginine, allantoin, polyphosphates, and purines have been reported to be sequestered, most likely in the cell vacuole. (19, 31, 33, 36, 37, 47, 50). This organelle, which has been suggested by Wiemken to be functionally related to lysosomes (25), has been reported to contain invertase, polyphosphate kinae, uricase, and all three major proteases (3, 24, 25, 31, 33). Consistent with the possible role of the vacuole maintaining internal homeostasis in the face of an adverse external environment are reports citing marked changes in vacuole morphology during cell division and sporulation (29, 36); the latter process occurs only under conditions of starvation. That turnover of previously synthesized cell constituents might also be an important source of internal nutrients is circumstantially supported by many reports. Betz and Weiser observed the loss of several specific enzyme activities during sporulation (6, 7), whereas Hopper et al. observed turnover of gross cellular protein and identified the times during sporulation when it was apparently degraded (18). Recently Bakalkin and his collaborators reported that protein degradation increased two- to threefold in late-log-phase cultures of S. cerevisiae (1, 2). Here we report experiments which indirectly monitored vacuole or vesicle permeability and protease involvement in the maintenance of internal homeostasis during external nutrient deprivation and treatment of a cells with a-factor. Our data suggest that both vacuole or vesicle permeability and proteolytic action are highly regulated and responsive to nutrient limitation and a-factor treatment. Another result of this work has been development of procedures which can be used to study the molecular events associated with commitment of a cell to initiate the cell division cycle even under experimental conditions in which division itself can not be completed. Several preliminary reports of this work have already appeared (R. Sumrada and T. G. Cooper, Fed. Proc. 36:917, 1977; Sumrada and Cooper, Abstr. Annu. Meet. Am. Soc. Microbiol.
235
1977, K244, p. 226; Sumrada and Cooper, Abstr. 1977 Meet. Mol. Biol. Yeast, 3). MATERIALS AND METHODS Strains. All of the strains used in this work have been described previously (23, 41, 44). Their genotypes and biochemical defects are summaed in Table 1. When haploid strains were used, the medium was supplemented with the metabolites they required for growth (20 mg/liter). For diploid strains supplementation was not necessary because they are all prototrophic. Culture conditions. The medium used throughout these experiments was that of Wickerham (45). Except where indicated otherwise, glucose (0.6%) and ammonia (0.1%) were added as sole sources of carbon and nitrogen, respectively. Several experiments were performed with low-sulfate and sulfate-free media. Sulfate-free medium contained (per liter of distilled water): KH2PO4, 0.2 g; Na3CsH507-2H20, 1.0 g; MgCl2.6H20, 0.2 g; Zn(C2HE02)2* 2H20, 4 mg; FeCl6-6H20, 0.5 mg; CuCl2-2H20, 0.26 mg; d-biotin, 0.1 mg; calcium pantothenate, 5.0 mg; inositol, 100 mg; thiamine, 60 mg; pyridoxine hydrochloride, 10 mg; ammonium chloride, 1.0 g; and glucose, 6.0 g. The final pH of the medium was 5.0. Low-sulfate medium contained 0.05 mM ammonium sulfate in addition to the above components. All cultures were maintained at
300C.
Cell density measurements. Turbidimetric measurements of cell density were made with a Klett-Summerson colorimeter (500- to 570-nm bandpass filter). One-hundred Klett units was approximately equivalent to 3 x 107 cells per ml of culture. Visual determination of cell numbers and the ratios of budded to unbudded cells were performed with a Petroff-Hauser counting chamber. Cell samples removed for counting purposes were fixed by adding an equal volume of solution containing 0.15 M NaCl and 3.7% formaldehyde. Cell aggregates were broken up by vigorous mixing with a Vortex mixer and by repeatedly drawing the solution into a Pasteur pipette and forcibly expelling it. This procedure was used in preference to sonic TABLE 1. Strains of S. cerevisiae used Enzyme defect Genotype DesJignation XT1172-S185 M63-ld M67-lOb M83-lb M61-17d M108-104d M25 M62 M58
M85
a ade6 kul a Iysl a lysl a lysl carl a Iysl durl a Iysl dall a ade6 eul
None None None
Arginase Urea carboxylase \
(a his6 ural Iysl) cta ase6 kul durl
(a hia6 ural durl aiade6 kul carl
(a his6 ural carl a ade6 leul dall a his6 ural dall
M319
a hu7 lys2 prtl a adel ural prti)
Allantoinase None
Urea carboxylase b
Arginase
Alltoinase ti
prtl gene productprotein synthesis inu'tiation
236
SUMRADA AND COOPER
oscillation, which produced some cell damage. However, we quantitatively compared ratios obtained by both methods and found no significant differences between them. For each value reported, 500 to 900 celis were counted. To eliminate subjective judgments concerning the point at which a budded cell was considered as two cells, budded cells were considered as ingle cells regardless of the bud size. Cell counts were completed within 48 h; subsequent to this time, cells appeared to become more fragile with age. Transfer of cells fom one medium to another. In a number of experiments it was necessary to transfer cell samples from one medium to another. This was done by filtering the culture through membrane filters and resuspending the harvested cells in fresh medium. All filtrations were performed in less than 15 to 20 s. The extent of cell loss during this procedure was determined by using radioactively labeled cells and was found to be negligible (8). Preparation of a-factor. The procedure for isolation of a-factor was that of Duntze et aL (14). Strain XT1172-S185 was grown to late log phase in 45 liters of Difco yeast nitrogen base medium supplemented with adenine and leucine. Glucose (2%) and ammonium sulfate (0.1%) were provided as sole carbon and nitrogen sources, respectively. The cultures were vigorously aerated during growth by compressed air forced through spargers. The medium was collected by centrifugation in a Sharples Ti centrifuge, cooled to 40C, and adsorbed (flow rate, 600 ml/h) onto a column (5 by 10 cm) of Amberlite CG 50 equilibrated with 0.1 N acetic acid. After complete adsorption of charged materials, 1 liter of 50% ethanol was passed through the column and discarded. One liter of 80% ethanol containing 0.01 N HCI was then used to elute a-factor. This fraction was collected and concentrated to about 200 ml with a Buchi rotary evaporator. Water (300 to 400 ml) was added, and the solution was again concentrated to 150 ml at 400C. This step was repeated six times to remove all traces of ethanol. The aqueous solution was frozen in dry ice-acetone and lyophilized. The light brown powder that resulted was dissolved in 50 ml of redistiled methanol, concentrated to 30 ml by evaporation, and stored at -20°C. Before a preparation of a-factor was used, its bio-
logical activity was asesed by adding increasing con-
centrations of the preparation to a sensitive haploid strain. The methanol solution was evaporated under a gentle stream of air before the cells were added. The amount of a-factor-containing solution which brought about complete arrest for 10 h (usually 10 to 20 pl/ml of culture) was used for our experiments. This method of assay has also been recommended by Stotzler et al. (35). Determination of allophanate hydrolase activity. Allophanate hydrolaw activity was measured by determination of evolved 14CO2 derived from ['4C]allophanate, using the methods of Whitney et al. (43). Asay of gross protein synthesis. Protein synthesis was measured by incorporation of [3H]leucine into hot trichloroacetic acid-precipitable material. Cultures were incubated with 15 pg of [8HJleucine (specific activity, 87 pCi/pumol) per ml. At the times indicated, 0.2-ml samples were removed from the culture and transferred to cold test tubes, followed im-
J. BACTERIOL.
mediately by addition of 4 ml of ice-cold 10% trichloroacetic acid containing 1 mg of nonradioactive leucine per ml. The samples were covered with marbles and placed in a boiling-water bath for 15 to 20 min. Precipitated protein was collected on nitrocellulose filters (0.45-pum pore diameter) and washed five times with 4 ml of cold 5% trichloroacetic acid. Filters were dried for 1 h at 800C to remove all traces of trichloroacetic acid, and the radioactivity they contained was determined with a standard toluene scintillation fluid.
RESULTS Turnover of allophanate hydrolase during normal growth. Studies of protein turnover can be performed in two ways. The first method is to follow total cellular protein. The experimental fonnat usually involves monitoring a radioactive amino acid in hot trichloroacetic acid-precipitable material. Although this method is often used, it has the disadvantage of measuring the "average" behavior of all proteins and has quite limited resolution. Another method is the assay of a specific enzyme. Although an immunochemical assay is most desirable, enzyme activity itself can at times provide a good tentative estimate of turnover. An important advantage offered by this approach is its sensitivity. A single cleavage is quite likely to render most enzymes inactive but may not give rise to acid-soluble material or detectably altered gel patterns of immunoprecipitates. Therefore, we chose allophanate hydrolase, the last enzyme of the aLlantoin degradative pathway, as our probe. Also, the genetics, biochemistry, and physiology of this enzyme have already been studied in great detail (8, 9, 22, 23, 41-43). Allophanate hydrolase does not appear to turn over during normal logarithmic growth. This was demonstrated by monitoring total enzyme activity for several generations in a wild-type culture of S. cerevisiae that had received a short pulse of induction. There was no decrease in the amount of activity observed per unit volume of culture during the two or more generations monitored in this experiment (Fig. 1). However, the amount of activity per cell decreased logarithmically (Fig. 1A), with a half-life of 165 min or somewhat longer than the 143-min doubling time of the culture (Fig. 1B). This discrepancy was accounted for by the basal rate of hydrolase production that curred in the absence of inducer (note the slight increase in total activity in Fig. 1A). This slight increase in activity did not result from incomplete removal of inducer because, upon transfer of the cells to inducerfree medium, the intracellular concentration of radioactive inducer dropped to undetectable levels before the first sample could be taken (i.e., by 30 s after removal of the cells from radioactive
F
w y
w
1o2840
j I I I I induced cultures for a variety of nutrients and 0 monitored the effects of these treatments on A hydrolase activity. The onset and degree of cell 120 . = the cycle arrest were monitored by determining _ loo 00 _ \ r ratio of budded to unbudded cells; for exponenm *n tially growing cultures, this ratio ranged from so0 0.6 to 0.8. The effects of ammonia deprivation \ z on a growing culture of S. cerevisiae is depicted _ _ in Fig. 3. At zero time, part of a late-log-phase =60 _ culture was transferred from our standard glu4 3 40 cose-ammonia medium to fresh, prewarmed pre_ O aerated medium containing the non-metaboliz2 20 > oable
JOURNAL OF BACTERIOLOGY, Oct. 1978, p. 234-246 0021-9193/78/0136-0234$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Control of Vacuole Permeability and Protein Degradation by the Cell Cycle Arrest Signal in Saccharomyces cerevisiae ROBERTA SUMRADA AND TERRANCE G. COOPER* Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received for publication 28 April 1978
Saccharomyces cerevisiae responds to deprivation of nutrients by arresting cell division at the unbudded Gl stage. Cells situated outside of Gl at the time of deprivation 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 afactor 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 a cells or a/a diploid strains were treated with a-factor. These data suggest that release of vacuolar constituents and protein turnover may be regulated by the Gl arrest signal. Many "higher" and "lower" eucaryotic organisms control their cell division cycle in response to the availability of organic and inorganic nutrients. Saccharomyces cerevisiae respond to starvation for glucose, ammonia, sulfate, phosphate, biotin, or potassium by arresting cell division at the unbudded Gl stage (20, 49). Johnston et al. observed that nutrient-deprived cells situated outside of Gl were able to complete their cell cycles (21). However, they neither initiated new division cycles nor completed any of the three known gene-controlled steps (cdc28, cdc4, or cdc7 gene product function) suggested by Reid and Hartwell to be among the earliest detectable events in division (30). It has been hypothesized that yeast celLs have some mechanism for monitoring the levels of required nutrients to ensure their presence before a new round of cell division is initiated (20). In an effort to identify the biochemical events asociated with monitoring sulfate sufficiency and the decision to "start" a round of division, Unger and Hartwell used mutants blocked in sulfate reduction, methionine biosynthesis, and methionyl-tRNA acylation (39). They concluded that the pivotal regulatory process occurred subsequent to the charging of tRNA. They could not proceed further to determine whether or not protein synthesis itself was involved in the de234
cision-making process of the cells, because loss of protein synthesis results in immediate cessation of growth and division irrespective of the position of the cells in the division cycle. Since their approach required that cells be able to complete division once they had successfuly passed the regulatory point, further progress with this experimental format was not possible. Although nutrient supply plays an important role in control of the cell cycle, it is by no means exclusive. S. cerevisiae have been shown to mate only when both mating cells are situated in Gl (10), which means that asynchronously growing a and a cells must synchronize their cell cycles before they can mate. This problem has apparently been solved by the elaboration of pheromones, which transiently arrest growth at Gl. Cells of the a mating type constitutively secrete a tridecapeptide hormone, a-factor, into the medium. Cells of the a mating type that come in contact with a-factor complete their cell division cycle and arrest as unbudded Gl cells. Arrest lasts for a period of time which is dependent, among other things, on the concentration of afactor encountered (12, 49). An analogous set of reciprocal events bring about Gl arrest of a celLs, although they have not yet been as well-studied biochemically (5, 12). The important feature of nutrient starvation
VOL. 136, 1978
CONTROL BY THE CELL CYCLE ARREST SIGNAL
and treatment with mating pheromones is that growth is arrested at the same point in both cases. This raises the possibility that some of the sensing and control elements may be shared in common, even though the two processes causing arrest are quite different. Our initial interest in the problem of G1 arrest was motivated by the question: How could cells complete division in the absence of sufficient external nutrients? We considered two possible nutrient sources that could be tapped during times of external deprivation: the cell vacuole and turnover of previously synthesized constituents. Major reserves of S-adenosylmethionine, arginine, allantoin, polyphosphates, and purines have been reported to be sequestered, most likely in the cell vacuole. (19, 31, 33, 36, 37, 47, 50). This organelle, which has been suggested by Wiemken to be functionally related to lysosomes (25), has been reported to contain invertase, polyphosphate kinae, uricase, and all three major proteases (3, 24, 25, 31, 33). Consistent with the possible role of the vacuole maintaining internal homeostasis in the face of an adverse external environment are reports citing marked changes in vacuole morphology during cell division and sporulation (29, 36); the latter process occurs only under conditions of starvation. That turnover of previously synthesized cell constituents might also be an important source of internal nutrients is circumstantially supported by many reports. Betz and Weiser observed the loss of several specific enzyme activities during sporulation (6, 7), whereas Hopper et al. observed turnover of gross cellular protein and identified the times during sporulation when it was apparently degraded (18). Recently Bakalkin and his collaborators reported that protein degradation increased two- to threefold in late-log-phase cultures of S. cerevisiae (1, 2). Here we report experiments which indirectly monitored vacuole or vesicle permeability and protease involvement in the maintenance of internal homeostasis during external nutrient deprivation and treatment of a cells with a-factor. Our data suggest that both vacuole or vesicle permeability and proteolytic action are highly regulated and responsive to nutrient limitation and a-factor treatment. Another result of this work has been development of procedures which can be used to study the molecular events associated with commitment of a cell to initiate the cell division cycle even under experimental conditions in which division itself can not be completed. Several preliminary reports of this work have already appeared (R. Sumrada and T. G. Cooper, Fed. Proc. 36:917, 1977; Sumrada and Cooper, Abstr. Annu. Meet. Am. Soc. Microbiol.
235
1977, K244, p. 226; Sumrada and Cooper, Abstr. 1977 Meet. Mol. Biol. Yeast, 3). MATERIALS AND METHODS Strains. All of the strains used in this work have been described previously (23, 41, 44). Their genotypes and biochemical defects are summaed in Table 1. When haploid strains were used, the medium was supplemented with the metabolites they required for growth (20 mg/liter). For diploid strains supplementation was not necessary because they are all prototrophic. Culture conditions. The medium used throughout these experiments was that of Wickerham (45). Except where indicated otherwise, glucose (0.6%) and ammonia (0.1%) were added as sole sources of carbon and nitrogen, respectively. Several experiments were performed with low-sulfate and sulfate-free media. Sulfate-free medium contained (per liter of distilled water): KH2PO4, 0.2 g; Na3CsH507-2H20, 1.0 g; MgCl2.6H20, 0.2 g; Zn(C2HE02)2* 2H20, 4 mg; FeCl6-6H20, 0.5 mg; CuCl2-2H20, 0.26 mg; d-biotin, 0.1 mg; calcium pantothenate, 5.0 mg; inositol, 100 mg; thiamine, 60 mg; pyridoxine hydrochloride, 10 mg; ammonium chloride, 1.0 g; and glucose, 6.0 g. The final pH of the medium was 5.0. Low-sulfate medium contained 0.05 mM ammonium sulfate in addition to the above components. All cultures were maintained at
300C.
Cell density measurements. Turbidimetric measurements of cell density were made with a Klett-Summerson colorimeter (500- to 570-nm bandpass filter). One-hundred Klett units was approximately equivalent to 3 x 107 cells per ml of culture. Visual determination of cell numbers and the ratios of budded to unbudded cells were performed with a Petroff-Hauser counting chamber. Cell samples removed for counting purposes were fixed by adding an equal volume of solution containing 0.15 M NaCl and 3.7% formaldehyde. Cell aggregates were broken up by vigorous mixing with a Vortex mixer and by repeatedly drawing the solution into a Pasteur pipette and forcibly expelling it. This procedure was used in preference to sonic TABLE 1. Strains of S. cerevisiae used Enzyme defect Genotype DesJignation XT1172-S185 M63-ld M67-lOb M83-lb M61-17d M108-104d M25 M62 M58
M85
a ade6 kul a Iysl a lysl a lysl carl a Iysl durl a Iysl dall a ade6 eul
None None None
Arginase Urea carboxylase \
(a his6 ural Iysl) cta ase6 kul durl
(a hia6 ural durl aiade6 kul carl
(a his6 ural carl a ade6 leul dall a his6 ural dall
M319
a hu7 lys2 prtl a adel ural prti)
Allantoinase None
Urea carboxylase b
Arginase
Alltoinase ti
prtl gene productprotein synthesis inu'tiation
236
SUMRADA AND COOPER
oscillation, which produced some cell damage. However, we quantitatively compared ratios obtained by both methods and found no significant differences between them. For each value reported, 500 to 900 celis were counted. To eliminate subjective judgments concerning the point at which a budded cell was considered as two cells, budded cells were considered as ingle cells regardless of the bud size. Cell counts were completed within 48 h; subsequent to this time, cells appeared to become more fragile with age. Transfer of cells fom one medium to another. In a number of experiments it was necessary to transfer cell samples from one medium to another. This was done by filtering the culture through membrane filters and resuspending the harvested cells in fresh medium. All filtrations were performed in less than 15 to 20 s. The extent of cell loss during this procedure was determined by using radioactively labeled cells and was found to be negligible (8). Preparation of a-factor. The procedure for isolation of a-factor was that of Duntze et aL (14). Strain XT1172-S185 was grown to late log phase in 45 liters of Difco yeast nitrogen base medium supplemented with adenine and leucine. Glucose (2%) and ammonium sulfate (0.1%) were provided as sole carbon and nitrogen sources, respectively. The cultures were vigorously aerated during growth by compressed air forced through spargers. The medium was collected by centrifugation in a Sharples Ti centrifuge, cooled to 40C, and adsorbed (flow rate, 600 ml/h) onto a column (5 by 10 cm) of Amberlite CG 50 equilibrated with 0.1 N acetic acid. After complete adsorption of charged materials, 1 liter of 50% ethanol was passed through the column and discarded. One liter of 80% ethanol containing 0.01 N HCI was then used to elute a-factor. This fraction was collected and concentrated to about 200 ml with a Buchi rotary evaporator. Water (300 to 400 ml) was added, and the solution was again concentrated to 150 ml at 400C. This step was repeated six times to remove all traces of ethanol. The aqueous solution was frozen in dry ice-acetone and lyophilized. The light brown powder that resulted was dissolved in 50 ml of redistiled methanol, concentrated to 30 ml by evaporation, and stored at -20°C. Before a preparation of a-factor was used, its bio-
logical activity was asesed by adding increasing con-
centrations of the preparation to a sensitive haploid strain. The methanol solution was evaporated under a gentle stream of air before the cells were added. The amount of a-factor-containing solution which brought about complete arrest for 10 h (usually 10 to 20 pl/ml of culture) was used for our experiments. This method of assay has also been recommended by Stotzler et al. (35). Determination of allophanate hydrolase activity. Allophanate hydrolaw activity was measured by determination of evolved 14CO2 derived from ['4C]allophanate, using the methods of Whitney et al. (43). Asay of gross protein synthesis. Protein synthesis was measured by incorporation of [3H]leucine into hot trichloroacetic acid-precipitable material. Cultures were incubated with 15 pg of [8HJleucine (specific activity, 87 pCi/pumol) per ml. At the times indicated, 0.2-ml samples were removed from the culture and transferred to cold test tubes, followed im-
J. BACTERIOL.
mediately by addition of 4 ml of ice-cold 10% trichloroacetic acid containing 1 mg of nonradioactive leucine per ml. The samples were covered with marbles and placed in a boiling-water bath for 15 to 20 min. Precipitated protein was collected on nitrocellulose filters (0.45-pum pore diameter) and washed five times with 4 ml of cold 5% trichloroacetic acid. Filters were dried for 1 h at 800C to remove all traces of trichloroacetic acid, and the radioactivity they contained was determined with a standard toluene scintillation fluid.
RESULTS Turnover of allophanate hydrolase during normal growth. Studies of protein turnover can be performed in two ways. The first method is to follow total cellular protein. The experimental fonnat usually involves monitoring a radioactive amino acid in hot trichloroacetic acid-precipitable material. Although this method is often used, it has the disadvantage of measuring the "average" behavior of all proteins and has quite limited resolution. Another method is the assay of a specific enzyme. Although an immunochemical assay is most desirable, enzyme activity itself can at times provide a good tentative estimate of turnover. An important advantage offered by this approach is its sensitivity. A single cleavage is quite likely to render most enzymes inactive but may not give rise to acid-soluble material or detectably altered gel patterns of immunoprecipitates. Therefore, we chose allophanate hydrolase, the last enzyme of the aLlantoin degradative pathway, as our probe. Also, the genetics, biochemistry, and physiology of this enzyme have already been studied in great detail (8, 9, 22, 23, 41-43). Allophanate hydrolase does not appear to turn over during normal logarithmic growth. This was demonstrated by monitoring total enzyme activity for several generations in a wild-type culture of S. cerevisiae that had received a short pulse of induction. There was no decrease in the amount of activity observed per unit volume of culture during the two or more generations monitored in this experiment (Fig. 1). However, the amount of activity per cell decreased logarithmically (Fig. 1A), with a half-life of 165 min or somewhat longer than the 143-min doubling time of the culture (Fig. 1B). This discrepancy was accounted for by the basal rate of hydrolase production that curred in the absence of inducer (note the slight increase in total activity in Fig. 1A). This slight increase in activity did not result from incomplete removal of inducer because, upon transfer of the cells to inducerfree medium, the intracellular concentration of radioactive inducer dropped to undetectable levels before the first sample could be taken (i.e., by 30 s after removal of the cells from radioactive
F
w y
w
1o2840
j I I I I induced cultures for a variety of nutrients and 0 monitored the effects of these treatments on A hydrolase activity. The onset and degree of cell 120 . = the cycle arrest were monitored by determining _ loo 00 _ \ r ratio of budded to unbudded cells; for exponenm *n tially growing cultures, this ratio ranged from so0 0.6 to 0.8. The effects of ammonia deprivation \ z on a growing culture of S. cerevisiae is depicted _ _ in Fig. 3. At zero time, part of a late-log-phase =60 _ culture was transferred from our standard glu4 3 40 cose-ammonia medium to fresh, prewarmed pre_ O aerated medium containing the non-metaboliz2 20 > oable
