JOURNAL OF BACTERIOLOGY, Mar. 1979, p. 1185-1190 0021-9193/79/03-1185/06$02.00/O
Vol. 137, No. 3
Synthesis and Modification of Proteins During the Cell Cycle of the Yeast Saccharomyces cerevisiae STEVEN G. ELLIOTT AND CALVIN S. McLAUGHLIN* Department of Biological Chemistry, College of Medicine, University of California, Irvine, Irvine, California 92717 Received for publication 11 September 1978
We have used a novel technique to study the synthesis, modification and degradation of proteins during the cell cycle in Saccharomyces cerevisiae. Logarithmically growing cells were pulse-labeled twice, with the pulses separated in time by more than one generation. Subsequently, the cells were fractionated as to their position in the cell cycle by centrifugal elutriation, and for different proteins the ratio of radioactive material from the two pulses was then -determined. Periodic degradation, synthesis, or modification would produce periodic variations in the ratio of counts. Two-dimensional gel electrophoresis was used to examine 110 different proteins at different times of the cell cycle. All but two proteins had a constant ratio of counts through the cell cycle. This indicates that the rate of synthesis of individual proteins increases exponentially during the cell cycle and that periodic degradation or modification of proteins is not a general feature of the cell cycle in S. cerevisiae.
The synthesis of total protein, total RNA (1), rRNA, tRNA, polyadenylic acid-containing RNA (manuscript in preparation), ribosomal proteins (manuscript in preparation), and cellular proteins (1) increases in an exponential manner during the cell cycle of the yeast Saccharomyces cerevisiae. Still, numerous studies indicate that the activities of many enzymes vary periodically during the cell cycle (3, 7). It now appears unlikely that all of these changes in activity are due to changes in synthesis. However, periodic modification and/or degradation of continuously synthesized proteins could play a role in the observed variations in enzyme activity. This report describes a novel dual-labeling method which can be used to study periodic degradation or modification of proteins during the cell cycle. A previous method involving continuous radioactive labeling of macromolecules followed by a short pulse has been useful in studying synthesis during the cell cycle (1). The method described here utilizes two pulses of radioactive precursors spaced more than a cell cycle apart in time in a logarithmically growing culture. If periodic degradation occurs, the first pulse label will fluctuate periodically through the cell cycle, whereas the second pulse label will be incorporated at an exponentially increasing rate. Assuming exponential synthesis, the ratio of the two pulses will show a peak or a step if periodic degradation occurs (Fig. 1D). The
magnitude of the increase in the ratio of the two pulses will reflect the change in rate of degradation. The start of the degradation period will match the increased ratio; however, the end of the increased ratio will depend on the time between pulses and the length of the degradation period. In the experimental regime used here, the time between pulses was 1.7 cell cycles. Therefore, there should be a drop in ratio of the pulses of approximately one-third of a cell cycle after the end of the degradation period. If there is no degradation and exponential synthesis occurs, the ratio will remain constant (at c) through the cell cycle (Fig. 1A). Periodic synthesis will have a positive ratio only during the synthetic period (Fig. 1B). Assuming no basal synthesis and perfect separation of the cell cycle, the variation in minimum to maximum ratio will approach infinity for this case. Practically, this variation in ratio will be limited by imperfections in the synchronizing procedure and time constraints imposed by the pulse length. In any case, a variation in ratio of 10-fold or greater would be expected. Step synthesis is constant linear synthesis with a doubling in rate of synthesis at a defined point in the cell cycle. This results in a stepwise increase and decrease in the ratio of the two pulses (Fig. 10). The ratio will double abruptly at the time of the rate doubling. It will exactly halve again after a period of time dependent on the time between pulses, which in this case would be approximately one-third of
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growth was allowed to continue for 3 h. Then 0.25 ACi of [3S]methionine/ml was added. After 10 min, growth was stopped by the addition of one-third volume of crushed ice to the medium. Cells were immediately pelleted, resuspended in distilled water, and held at 4°C throughout the rest of the experiment. Cell cycle fractionation. The basic procedure used for cell cycle fractionation has been described previously (2). However, several imnprovements have been imposed. Separation was performed in a chamber designed by Sanderson (12) and generously provided by Beckman Instruments, Inc. A manostat flow meter (V.W.R.) was used to measure flow rate, and a bubble chamber, provided by Beckman Instruments, was interposed between the rotor and the pump. The bubble chamber had a capacity of 100 ml and was filed twothirds full of water. A bypass valve allowed flow through the chamber containing the air pocket, which dampened the surging effect caused by the pump. This resulted in a smoother flow of water through the rotor chamber. For rapid loading of the sample, the bypass valve was positioned that flow of liquid was directly from the sample reservoir to the rotor. After loading of the sample, the bypass valve was repositioned to allow flow through the bubble chamber. After 5 min of stabilization, the culture was fractionated normally (2). The first fraction was collected at 11 ml/min, and successive fractions were collected by increasing the flow rate in increments of 2 ml/min. To examine the quality of separation, cells were fixed with formaldehyde and stained with Giemsa, and the proportion of specified cell types in each fraction was determined microscopically (1, 5). Two-dimensional gel electrophoresis. Amounts of 5 id of RNase-DNase solution were added to cell pellets (approximately 5 x 10' cells) at 0 to 4°C in 1ml glass tubes (1). Glass beads, 0.45 mm (Glasperlen), were added to the meniscus. The tubes were blended in a Vortex mixer four times for 30 s, and 5 tLd of sodium dodecyl sulfate (SDS) lysis buffer (1) was then added. Immediately prior to electrophoresis, solid urea was added to a concentration of 9.5 M followed by 10 pl of sample buffer (1) containing 4% ampholytes (0.8%, pH 3 to 10; 1.6%, pH 5 to 8; 1.6%, pH 5 to 7). Electrophoresis was then performed as described previously (1, 10). so
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FIG. 1. Theoretical early pulse-labeling to late pulse-labeling ratios for four models of accumulation or degradation. (A) Exponentially increasing rate of synthesis; (B) periodic synthesis; (C) step synthesis (a doubling in the rate of synthesis at a certain point in the cell cycle); (D) periodic degradation.
the cell cycle. Combination of this dual-label technique with O'Farrell two-dimensional polyacrylamide gel electrophoresis allows an analysis of synthesis and degradation of individual proteins during the cell cycle. MATERIAILS AND METHODS Strains. A diploid of S. cerevisiae with the classification SKQ2n was used in all the experiments. It was obtained from Brian Cox (University of Oxford) and has the genotype a/a adel/+ +/adel +/hisl. Chemicals. Electrophoresi-grade acrylamide, N,N'-methylene bisacrylamide, and N,N,N',N'-tetramethylenediamine were from Eastman Kodak. Ampholines were from L.K.B. Ultrapure urea and [3H]methionine (500 mCi/mmol) were from Schwarz/ Mann. [3S]methionine (500 to 800 Ci/mmol) was from Amersham Corp. All other chemicals were from standard commercial sources. Growth and labeling of cells. Proteins were labeled by a modified dual-label procedure in medium containing, per liter, 6.7 g of yeast nitrogen base minus amino acids (Difco), 12.5 mg each of all the amino acids except methionine and cysteine, 20 g of glucose, 20 mg of adenine, and 20 mg of uracil. Cells were grown to midlog phase on a rotary shaker at 230C. [3H]methionine (10 ACi/ml) was added and
RESULTS Cells were grown and fractionated by centrifugal elutriation, with the use of the improvements described in Materials and Methods. Growth in YM-1 medium (4) gave similar results. Each fraction was stained with Giemsa, and the proportion of each cell type, shown schematically in the inset in Fig. 2, was determined. Cell cycle landmarks were well resolved. Each cell type reached a purity of at least 50%. Each cell type peaked and then dropped to a low level (5 to 10%). When the total number of cell types was summed and compared to logphase cells, the proportion of budded mononucleate celLs and mitotic cells was the same. However, in fractionated cells, unbudded cells had
REGULATION OF PROTEINS DURING THE CELL CYCLE
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longer exposure brought out additional fainter spots. Approximately 60% of the counts entered the gel, and 7% of the total was present in the
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(ml /min) FIG. 2. Separation of yeast cell markers by centrifugal elutriation. The inset shows four different cell types examined in fractions obtained at the indicated flow rates. (a) Unbudded cells; (A) budded cells with a single nucleus; (5) cells with migrating nuclei; (0) binucleate cells. Cells were stained with Giemsa, and the proportion of each cell type in each fraction was determined microscopically.
increased by 5% and binucleate cells had decreased by 5%, indicating that binucleate cells were converted to mononucleate cells. This was a result of mechanical breakage caused by subsequent sample manipulation and not a result of growth. If growth were occurring, there would be no change in the proportion of the various cell types. The 5% change explains the residual amount of unbudded cells (6%) observed in late fractions as well as the sharp increase in unbudded cells observed at 29 ml/min. Two-dimensional O'Farrell gels (10) were used to assay the rate of synthesis of individual proteins. A map of a typical two-dimensional gel autoradiogram of total yeast protein labeled with [35S]methionine, prepared and subjected to electrophoresis as described in Materials and Methods, is shown in Fig. 3. Isoelectric focusing is left to right, with the basic end on the left. The pH range is approximately 7.5 to 4.2. SDS electrophoresis is from top to bottom. The 1-cm marks placed along the edges allow an estimate of relative positions. Over 550 different protein spots were detectable on the gel represented in Fig. 3. In this case the autoradiogram was developed after 5 days;
vertical streak at the basic end of the gel on the left. The streak was attributed to basic and insoluble proteins that precipitated shortly after entering the gel and to sulfur-containing carbohydrate. Individual protein spots which were analyzed were given the numbers indicated on the map. To determine the molecular weight range of proteins on the two-dimensional gels, we subjected proteins of known molecular weight to electrophoresis in the same manner, and their positions in the SDS dimension are shown in Fig. 3. Extrapolation indicates that proteins with molecular weights between 12,500 and 155,000 will run in this system. To determine whether proteins were synthesized or degraded periodically, logarithmically growing cells were labeled by the dual-pulse method and were separated into fractions representing different positions in the cell cycle. Samples from each fraction were subjected to electrophoresis as described in Materials and Methods. The first pulse was [3H]methionine and the second was [3S]methionine. Virtually all of the first pulse was incorporated within 20 min. After 20 min, the radioactivity stabilized and remained constant throughout the experiment. The ratio of radioactive material in individual spots was then determined. A plot of ratio versus flow rate for protein 67 (Fig. 3) is depicted in Figure 4. The ratio for this protein remained constant through the cell cycle at a value of 0.24, indicating that its rate of synthesis increased exponentially during the cell cycle and that it was not degraded periodically. Altogether, 110 different spots were analyzed by the pulse-pulse dual-label method, including most of the ones examined before by the longterm dual-label method (1). The ratios of new pulse radioactivity divided by old pulse radioactivity for 50 of the 110 proteins analyzed are shown in Table 1. All except 2 (proteins 24 and 209) of the 50 showed a constant ratio. The exceptions both had a higher than normal ratio, which is indicative of increased degradation. Of the 110 proteins examined by this method, 108 showed a constant ratio. This indicates that most of these proteins are synthesized in an exponential manner and are not degraded periodically. DISCUSSION The mechanism of accumulation of macromolecules during the cell cycle has been studied for some time. However, whether variations in the activity of synthesis of proteins do or do not
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containing 10i cpm was subjected to electrophoresis as described in Materials and Methods. After a 5-day exposure, the autoradiogram was developed by standard procedures (1), and the map shown was prepared. IF, isoelectric focusing; SDS, sodium dodecyl sulfate electrophoresis. Marks along the edges represent I cm and are included to establish relative positions. Numbered proteins are those that were analyzed. Molecular weight markers were bovine serum albumin (67,000), ovalbumin (45,000), DNase 1 (31,000), and chymotrypsinogen (25,100). (Reprinted, with permission, from reference 1)
has been the subject of much controversy (3, 7, 8). S. cerevisiae has many advantages for cell cycle studies of this type. It is one of the few eucaryotic experimental organisms that allows study by a combined genetic and biochemical approach. Furthermore, size selection can provide fractions containing cells in different positions in the cell cycle (2, 13). Labeling of cells followed by size selection will therefore allow an analysis on cells that have been unperturbed. Finally, the position in the cell cycle can be determined rapidly by simple cytological and biochemical tests (6, 7). The size selection scheme used here was centrifugal elutriation (2, 11). It compares favorably with other methods of this type (2). The improvements described here generate a separation of cell types 50 to 100% better than that described previously (1, 2). Therefore, any periodicities in rate of degradation or synthesis will be well resolved. Dual-label experiments are especially sensitive to changes in rates of synthesis. In previous studies, use of a continuous long-term label followed by a short pulse label has been shown to occur
distinguish easily between periodic synthesis (DNA) and exponential synthesis (RNA and protein) (1). Models of accumulation during the cell cycle, other than exponential accumulation, predict a twofold or greater variation in ratio of counts during the cell cycle (1). Using the pulsepulse labeling method, we analyzed 110 different protein spots. In all but 2 of the 110 spots, new pulse counts divided by old pulse counts remained constant, with some minor variation through the cell cycle. However, the variation was much less than the twofold changes in ratio predicted by mechanisms other than exponential synthesis. This means that the apparent rate of synthesis increases exponentially during the cell cycle. Methionine was used for pulse labeling, so the apparent rate approximates the true rate. That is, pool corrections are negligible because the pool for methionine is half-saturated within 20 s (14). Furthermore, the specific activity of the methionine pool remains constant during the cell cycle (manuscript in preparation). The unusually high average ratios observed for proteins 24 and 209, 1.03 and 2.26, respectively, versus an
VOL. 137, 1979 13.
REGULATION OF PROTEINS DURING THE CELL CYCLE FLOW RATE (mllmin) 17 19 21 23
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protein complexes, or monomer-dimer changes, would not be detectable since the conditions of electrophoresis result in completely denatured proteins. This work confirms the results of a previous study which used a long-term label followed by a short pulse label (1). In that study, it was
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FIG. 4. Ratio ofpulse radioactivities of a protein through the cell cycle. Protein was dual-labeled and fractionated by elutriation. Electrophoresis was performed on each fraction. For each fraction, spot 67 (Fig. 3) was cut out and the ratio of radioactivites in it was determined. For explanation of the assignment of cell cycle position, see reference 1.
of 0.36 for all 110 spots, are indicative of either increased degradation or modification. These same two proteins were examined by the long-term/pulse dual-label method and neither showed any periodic variations in ratio (1). Thus, it is unlikely that the periodicity observed here is due to periodic synthesis or step synthesis. This suggests that the variations in these two proteins are probably due to periodic degradation or modification. The pulse-pulse labeling method described here can distinguish between various patterns of protein accumulation. Since the rate of synthesis through the cell cycle is computed from the ratio of two pulse labels separated in time, degradation and some types of protein modifications can be detected. If new and old proteins are modified or degraded differentially, then the 35S/3H ratio will change as if there was a sudden increase or decrease in the rate of synthesis of the protein. Any periodic modification that alters the electrophoretic mobility of the polypeptide in either dimension would also result in a change in ratio. Since, in general, no changes were observed, we suggest that neither periodic modification that alters the position of two-dimensional gels nor periodic degradation is a general characteristic of the yeast cell cycle. However, some types of modifications would not be detected by this method. These would include those modifications that do not result in a charge change, e.g., average
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17 13 15 19 21 23 25 27 1 0.12 0.13 0.13 0.09 0.12 0.10 0.10 0.12 3 0.43 0.39 0.42 0.30 0.31 0.35 0.30 0.32 7 0.70 0.82 0.69 0.61 0.75 0.81 0.79 0.90 9 0.27 0.28 0.27 0.28 0.29 0.20 0.26 0.26 10 0.47 0.33 0.43 0.34 0.30 0.32 0.33 0.26 12 0.13 0.12 0.13 0.15 0.13 0.17 0.14 0.11 24 2.15 1.23 1.63 0.96 0.60 0.21 0.64 0.78 36 0.27 0.31 0.30 0.26 0.32 0.25 0.23 0.29 39 0.62 0.65 0.66 0.62 0.72 0.74 0.74 0.75 43 0.19 0.18 0.16 0.16 0.16 0.15 0.16 0.14 45 0.34 0.29 0.29 0.29 0.28 0.22 0.27 0.27 47 0.28 0.23 0.25 0.23 0.31 0.23 0.24 0.24 61 0.10 0.13 0.12 0.12 0.10 0.12 0.11 0.12 63 0.11 0.12 0.10 0.12 0.10 0.13 0.13 0.06 67 0.27 0.26 0.23 0.23 0.24 0.25 0.23 0.22 68 0.53 0.46 0.41 0.47 0.51 0.38 0.52 0.34 79 0.22 0.21 0.20 0.21 0.20 0.21 0.19 0.18 85 0.25 0.25 0.24 0.24 0.22 0.22 0.21 0.20 88 0.64 0.55 0.57 0.61 0.58 0.58 0.61 0.60 144 0.22 0.22 0.17 0.19 0.23 0.31 0.19 0.18 146 0.23 0.25 0.26 0.26 0.27 0.26 0.26 0.26 161 0.39 0.27 0.35 0.32 0.29 0.40 0.32 0.32 163 0.40 0.39 0.43 0.35 0.40 0.42 0.36 0.27 167 0.29 0.31 0.38 0.35 0.35 0.21 0.26 0.27 169 0.18 0.22 0.16 0.18 0.19 0.19 0.16 0.18 171 0.19 0.16 0.17 0.13 0.24 0.21 0.15 0.15 182 0.26 0.28 0.23 0.25 0.20 0.35 0.31 0.22 183 0.19 0.17 0.18 0.17 0.17 0.17 0.16 0.15 184 0.24 0.24 0.22 0.26 0.24 0.26 0.21 0.22 186 0.29 0.23 0.23 0.23 0.27 0.22 0.22 0.25 193 0.21 0.20 0.19 0.21 0.17 0.22 0.20 0.20 200a 0.21 0.20 0.20 0.20 0.19 0.19 0.18 0.18 208 0.33 0.27 0.23 0.27 0.26 0.28 0.23 0.29 209 3.00 2.69 3.39 2.11 2.50 1.54 1.71 1.13 217 0.26 0.22 0.21 0.23 0.23 0.26 0.23 0.28 225 0.24 0.33 0.27 0.23 0.24 0.31 0.22 0.28 227 0.25 0.19 0.18 0.17 0.21 0.19 0.19 0.18 229 0.58 0.48 0.48 0.49 0.45 0.48 0.47 0.48 231 0.20 0.20 0.19 0.17 0.18 0.19 0.17 0.16 232 0.16 0.15 0.15 0.15 0.16 0.15 0.14 0.13 236 0.23 0.22 0.24 0.21 0.23 0.23 0.27 0.24 241 0.39 0.31 0.31 0.32 0.31 0.32 0.30 0.27 243 0.32 0.29 0.66 0.26 0.25 0.18 0.27 0.29 251 0.21 0.20 0.19 0.19 0.20 0.19 0.18 0.15 252 0.55 0.45 0.49 0.43 0.45 0.30 0.43 0.46 253 0.29 0.24 0.24 0.24 0.25 0.25 0.23 0.22 254 0.29 0.26 0.24 0.21 0.26 0.24 0.20 0.20 255 0.31 0.28 0.26 0.27 0.25 0.26 0.23 0.25 282 0.43 0.38 0.34 0.33 0.26 0.44 0.34 0.45 287 0.34 0.41 0.26 0.29 0.28 0.29 0.29 0.29 a The relationship between flow rate and cell cycle position can be deduced from Fig. 4.
J. BACTERIOL. 1190 ELLIOTT AND McLAUGHLIN shown that 111 different protein spots have an modification through the cell cycle compels a exponentially increasing rate of synthesis during rethinking of the present models controlling cell the cell cycle. Including the 110 proteins ana- division. lyzed here, 130 different polypeptide spots, numACKNOWLEDGMENTS bered in Fig. 3, were analyzed, and all showed were supported by Public Health SerThese investigations of the rate of Ninety an exponential synthesis. CA10628 awarded by the National Cancer Institute. 130 were tested by both the long-term label- viceWegrant thank Riley and Anita Newman for helpful discussions. pulse (1) and the pulse-pulse method. Since these 130 were selected randomly from the over LITERATURE CITED 550 spots that are detectable on autoradiograns S. and C. S. McLaughlin. 1978. Rate of G., 1. Elliott, of two-dimensional gels, we suggest that periodic macromolecular synthesis through the cell cycle of the variation in the rate of synthesis is not a general yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. characteristic of the cell cycle. U.S.A. 75:4384-4388. of Clearly, our results establish that for most 2. Gordon, C. N., and S. G. Elliott 1977. Fractionation Saccharomyces cerevisiae cell populations by centrifuproteins there is little or no periodic modificagal elutriation. J. Bacteriol. 129:97-100. tion, degradation, or change in the rate of syn- 3. Halvorson, H. O., B. L A. Carter, and P. Tauro. 1971. thesis during the cell cycle. However, our system Synthesis of enzymes during the cell cycle. Adv. Microb. Physiol. 6:47-106. does not resolve the basic and acidic proteins, L H. 1967. Macromolecule synthesis in temand the less abundant class of proteins was not 4. Hartwell, perature-sensitive mutants of yeast. J. Bacteriol. 93: analyzed. Regulatory proteins may exhibit pe1662-1670. riodic behavior since they are present often in 5. Hartwell, L H. 1970. Periodic density fluctuations during the yeast cell cycle and the selection of synchronous amounts of only a few copies per cell. In fact, cultures. J. Bacteriol. 104:1280-1285. periodic synthesis has been demonstrated for 6. Hartwell, L H., J. Culotti, J. Pringle, and B. Reid. in our system (9). histones, which do not run 1974. Genetic control of the cell division cycle in yeast. Whether our results can accommodate the nuScience 183:46-51. merous observations on the periodic nature of 7. Mitchison, J. M. 1969. Enzyme synthesis in synchronous cultures. Science 166:657-663. enzyme activity during the cell cycle (3, 7) is J. M. 1971. The biology of the cell cycle. uncertain. Many of these observations might in 8. Mitchison, University Press, Cambridge, U.K. fact be due to variations in enzyme activity. 9. Moll, R., and E. Wintersberger. 1976. Synthesis of yeast histones in the cell cycle. Proc. Natl. Acad. Sci. U.S.A. A number of general theories on the nature of 73:1863-1867. control of periodic synthesis, such as gene dou- 10. O'Farrell, P. H. 1975. High resolution two-dimensional bling, oscillatory repression, and linear reading, electrophoresis of proteins. J. Biol. Chem. 250:4007have been proposed to account for the observa4021. tions on the periodic nature of proteins. How- 11. Sanderson, R. J., and K. E. Bird. 1977. Cell separations by counterflow centrifugation. Methods Cell Biol. 15:1ever, these models appear unlikely for the ma14. jority of the proteins in S. cerevisiae. Doubling 12. Sanderson, R. I., K. E. Bird, N. F. Palmer, and J. in DNA has been shown to have no effect on the Brenman. 1976. Design principles for a counterflow centrifugation cell separation chamber. Anal. Biochem. rate of synthesis of other molecules, including 71:615-622. total protein, total RNA, double-stranded RNA, 13. Sebastian, J., B. L A. Carter, and H. 0. Halvorson. rRNA, tRNA, polyadenylic acid-containing 1971. Use of yeast populations fractionated by zonal RNA, and ribosomal proteins (1; manuscripts in centrifugation to study the cell cycle. J. Bacteriol. 108: 1045-1050. preparation). Therefore, it appears that the cell J. R., S. A. Morgan, and R. W. Shumnan. does not immediately use the additional DNA 14. Warner, 1976. Kinetics of labeling of the S-adenosylmethionine made during S phase. Continuous synthesis of pool of Saccharomyces cerevisiae. J. Bacteriol. 125: proteins with no periodic degradation and/or 887-891.
Vol. 137, No. 3
Synthesis and Modification of Proteins During the Cell Cycle of the Yeast Saccharomyces cerevisiae STEVEN G. ELLIOTT AND CALVIN S. McLAUGHLIN* Department of Biological Chemistry, College of Medicine, University of California, Irvine, Irvine, California 92717 Received for publication 11 September 1978
We have used a novel technique to study the synthesis, modification and degradation of proteins during the cell cycle in Saccharomyces cerevisiae. Logarithmically growing cells were pulse-labeled twice, with the pulses separated in time by more than one generation. Subsequently, the cells were fractionated as to their position in the cell cycle by centrifugal elutriation, and for different proteins the ratio of radioactive material from the two pulses was then -determined. Periodic degradation, synthesis, or modification would produce periodic variations in the ratio of counts. Two-dimensional gel electrophoresis was used to examine 110 different proteins at different times of the cell cycle. All but two proteins had a constant ratio of counts through the cell cycle. This indicates that the rate of synthesis of individual proteins increases exponentially during the cell cycle and that periodic degradation or modification of proteins is not a general feature of the cell cycle in S. cerevisiae.
The synthesis of total protein, total RNA (1), rRNA, tRNA, polyadenylic acid-containing RNA (manuscript in preparation), ribosomal proteins (manuscript in preparation), and cellular proteins (1) increases in an exponential manner during the cell cycle of the yeast Saccharomyces cerevisiae. Still, numerous studies indicate that the activities of many enzymes vary periodically during the cell cycle (3, 7). It now appears unlikely that all of these changes in activity are due to changes in synthesis. However, periodic modification and/or degradation of continuously synthesized proteins could play a role in the observed variations in enzyme activity. This report describes a novel dual-labeling method which can be used to study periodic degradation or modification of proteins during the cell cycle. A previous method involving continuous radioactive labeling of macromolecules followed by a short pulse has been useful in studying synthesis during the cell cycle (1). The method described here utilizes two pulses of radioactive precursors spaced more than a cell cycle apart in time in a logarithmically growing culture. If periodic degradation occurs, the first pulse label will fluctuate periodically through the cell cycle, whereas the second pulse label will be incorporated at an exponentially increasing rate. Assuming exponential synthesis, the ratio of the two pulses will show a peak or a step if periodic degradation occurs (Fig. 1D). The
magnitude of the increase in the ratio of the two pulses will reflect the change in rate of degradation. The start of the degradation period will match the increased ratio; however, the end of the increased ratio will depend on the time between pulses and the length of the degradation period. In the experimental regime used here, the time between pulses was 1.7 cell cycles. Therefore, there should be a drop in ratio of the pulses of approximately one-third of a cell cycle after the end of the degradation period. If there is no degradation and exponential synthesis occurs, the ratio will remain constant (at c) through the cell cycle (Fig. 1A). Periodic synthesis will have a positive ratio only during the synthetic period (Fig. 1B). Assuming no basal synthesis and perfect separation of the cell cycle, the variation in minimum to maximum ratio will approach infinity for this case. Practically, this variation in ratio will be limited by imperfections in the synchronizing procedure and time constraints imposed by the pulse length. In any case, a variation in ratio of 10-fold or greater would be expected. Step synthesis is constant linear synthesis with a doubling in rate of synthesis at a defined point in the cell cycle. This results in a stepwise increase and decrease in the ratio of the two pulses (Fig. 10). The ratio will double abruptly at the time of the rate doubling. It will exactly halve again after a period of time dependent on the time between pulses, which in this case would be approximately one-third of
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growth was allowed to continue for 3 h. Then 0.25 ACi of [3S]methionine/ml was added. After 10 min, growth was stopped by the addition of one-third volume of crushed ice to the medium. Cells were immediately pelleted, resuspended in distilled water, and held at 4°C throughout the rest of the experiment. Cell cycle fractionation. The basic procedure used for cell cycle fractionation has been described previously (2). However, several imnprovements have been imposed. Separation was performed in a chamber designed by Sanderson (12) and generously provided by Beckman Instruments, Inc. A manostat flow meter (V.W.R.) was used to measure flow rate, and a bubble chamber, provided by Beckman Instruments, was interposed between the rotor and the pump. The bubble chamber had a capacity of 100 ml and was filed twothirds full of water. A bypass valve allowed flow through the chamber containing the air pocket, which dampened the surging effect caused by the pump. This resulted in a smoother flow of water through the rotor chamber. For rapid loading of the sample, the bypass valve was positioned that flow of liquid was directly from the sample reservoir to the rotor. After loading of the sample, the bypass valve was repositioned to allow flow through the bubble chamber. After 5 min of stabilization, the culture was fractionated normally (2). The first fraction was collected at 11 ml/min, and successive fractions were collected by increasing the flow rate in increments of 2 ml/min. To examine the quality of separation, cells were fixed with formaldehyde and stained with Giemsa, and the proportion of specified cell types in each fraction was determined microscopically (1, 5). Two-dimensional gel electrophoresis. Amounts of 5 id of RNase-DNase solution were added to cell pellets (approximately 5 x 10' cells) at 0 to 4°C in 1ml glass tubes (1). Glass beads, 0.45 mm (Glasperlen), were added to the meniscus. The tubes were blended in a Vortex mixer four times for 30 s, and 5 tLd of sodium dodecyl sulfate (SDS) lysis buffer (1) was then added. Immediately prior to electrophoresis, solid urea was added to a concentration of 9.5 M followed by 10 pl of sample buffer (1) containing 4% ampholytes (0.8%, pH 3 to 10; 1.6%, pH 5 to 8; 1.6%, pH 5 to 7). Electrophoresis was then performed as described previously (1, 10). so
I
m
0.5
0
Cell
cycle
position
FIG. 1. Theoretical early pulse-labeling to late pulse-labeling ratios for four models of accumulation or degradation. (A) Exponentially increasing rate of synthesis; (B) periodic synthesis; (C) step synthesis (a doubling in the rate of synthesis at a certain point in the cell cycle); (D) periodic degradation.
the cell cycle. Combination of this dual-label technique with O'Farrell two-dimensional polyacrylamide gel electrophoresis allows an analysis of synthesis and degradation of individual proteins during the cell cycle. MATERIAILS AND METHODS Strains. A diploid of S. cerevisiae with the classification SKQ2n was used in all the experiments. It was obtained from Brian Cox (University of Oxford) and has the genotype a/a adel/+ +/adel +/hisl. Chemicals. Electrophoresi-grade acrylamide, N,N'-methylene bisacrylamide, and N,N,N',N'-tetramethylenediamine were from Eastman Kodak. Ampholines were from L.K.B. Ultrapure urea and [3H]methionine (500 mCi/mmol) were from Schwarz/ Mann. [3S]methionine (500 to 800 Ci/mmol) was from Amersham Corp. All other chemicals were from standard commercial sources. Growth and labeling of cells. Proteins were labeled by a modified dual-label procedure in medium containing, per liter, 6.7 g of yeast nitrogen base minus amino acids (Difco), 12.5 mg each of all the amino acids except methionine and cysteine, 20 g of glucose, 20 mg of adenine, and 20 mg of uracil. Cells were grown to midlog phase on a rotary shaker at 230C. [3H]methionine (10 ACi/ml) was added and
RESULTS Cells were grown and fractionated by centrifugal elutriation, with the use of the improvements described in Materials and Methods. Growth in YM-1 medium (4) gave similar results. Each fraction was stained with Giemsa, and the proportion of each cell type, shown schematically in the inset in Fig. 2, was determined. Cell cycle landmarks were well resolved. Each cell type reached a purity of at least 50%. Each cell type peaked and then dropped to a low level (5 to 10%). When the total number of cell types was summed and compared to logphase cells, the proportion of budded mononucleate celLs and mitotic cells was the same. However, in fractionated cells, unbudded cells had
REGULATION OF PROTEINS DURING THE CELL CYCLE
VOL. 137, 1979
1187
longer exposure brought out additional fainter spots. Approximately 60% of the counts entered the gel, and 7% of the total was present in the
z
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0
11
13
15
17 19 21 FLOW RATE
23
25
27
29
(ml /min) FIG. 2. Separation of yeast cell markers by centrifugal elutriation. The inset shows four different cell types examined in fractions obtained at the indicated flow rates. (a) Unbudded cells; (A) budded cells with a single nucleus; (5) cells with migrating nuclei; (0) binucleate cells. Cells were stained with Giemsa, and the proportion of each cell type in each fraction was determined microscopically.
increased by 5% and binucleate cells had decreased by 5%, indicating that binucleate cells were converted to mononucleate cells. This was a result of mechanical breakage caused by subsequent sample manipulation and not a result of growth. If growth were occurring, there would be no change in the proportion of the various cell types. The 5% change explains the residual amount of unbudded cells (6%) observed in late fractions as well as the sharp increase in unbudded cells observed at 29 ml/min. Two-dimensional O'Farrell gels (10) were used to assay the rate of synthesis of individual proteins. A map of a typical two-dimensional gel autoradiogram of total yeast protein labeled with [35S]methionine, prepared and subjected to electrophoresis as described in Materials and Methods, is shown in Fig. 3. Isoelectric focusing is left to right, with the basic end on the left. The pH range is approximately 7.5 to 4.2. SDS electrophoresis is from top to bottom. The 1-cm marks placed along the edges allow an estimate of relative positions. Over 550 different protein spots were detectable on the gel represented in Fig. 3. In this case the autoradiogram was developed after 5 days;
vertical streak at the basic end of the gel on the left. The streak was attributed to basic and insoluble proteins that precipitated shortly after entering the gel and to sulfur-containing carbohydrate. Individual protein spots which were analyzed were given the numbers indicated on the map. To determine the molecular weight range of proteins on the two-dimensional gels, we subjected proteins of known molecular weight to electrophoresis in the same manner, and their positions in the SDS dimension are shown in Fig. 3. Extrapolation indicates that proteins with molecular weights between 12,500 and 155,000 will run in this system. To determine whether proteins were synthesized or degraded periodically, logarithmically growing cells were labeled by the dual-pulse method and were separated into fractions representing different positions in the cell cycle. Samples from each fraction were subjected to electrophoresis as described in Materials and Methods. The first pulse was [3H]methionine and the second was [3S]methionine. Virtually all of the first pulse was incorporated within 20 min. After 20 min, the radioactivity stabilized and remained constant throughout the experiment. The ratio of radioactive material in individual spots was then determined. A plot of ratio versus flow rate for protein 67 (Fig. 3) is depicted in Figure 4. The ratio for this protein remained constant through the cell cycle at a value of 0.24, indicating that its rate of synthesis increased exponentially during the cell cycle and that it was not degraded periodically. Altogether, 110 different spots were analyzed by the pulse-pulse dual-label method, including most of the ones examined before by the longterm dual-label method (1). The ratios of new pulse radioactivity divided by old pulse radioactivity for 50 of the 110 proteins analyzed are shown in Table 1. All except 2 (proteins 24 and 209) of the 50 showed a constant ratio. The exceptions both had a higher than normal ratio, which is indicative of increased degradation. Of the 110 proteins examined by this method, 108 showed a constant ratio. This indicates that most of these proteins are synthesized in an exponential manner and are not degraded periodically. DISCUSSION The mechanism of accumulation of macromolecules during the cell cycle has been studied for some time. However, whether variations in the activity of synthesis of proteins do or do not
1188
J. BACTERIOL.
ELLIOTT AND McLAUGHLIN
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containing 10i cpm was subjected to electrophoresis as described in Materials and Methods. After a 5-day exposure, the autoradiogram was developed by standard procedures (1), and the map shown was prepared. IF, isoelectric focusing; SDS, sodium dodecyl sulfate electrophoresis. Marks along the edges represent I cm and are included to establish relative positions. Numbered proteins are those that were analyzed. Molecular weight markers were bovine serum albumin (67,000), ovalbumin (45,000), DNase 1 (31,000), and chymotrypsinogen (25,100). (Reprinted, with permission, from reference 1)
has been the subject of much controversy (3, 7, 8). S. cerevisiae has many advantages for cell cycle studies of this type. It is one of the few eucaryotic experimental organisms that allows study by a combined genetic and biochemical approach. Furthermore, size selection can provide fractions containing cells in different positions in the cell cycle (2, 13). Labeling of cells followed by size selection will therefore allow an analysis on cells that have been unperturbed. Finally, the position in the cell cycle can be determined rapidly by simple cytological and biochemical tests (6, 7). The size selection scheme used here was centrifugal elutriation (2, 11). It compares favorably with other methods of this type (2). The improvements described here generate a separation of cell types 50 to 100% better than that described previously (1, 2). Therefore, any periodicities in rate of degradation or synthesis will be well resolved. Dual-label experiments are especially sensitive to changes in rates of synthesis. In previous studies, use of a continuous long-term label followed by a short pulse label has been shown to occur
distinguish easily between periodic synthesis (DNA) and exponential synthesis (RNA and protein) (1). Models of accumulation during the cell cycle, other than exponential accumulation, predict a twofold or greater variation in ratio of counts during the cell cycle (1). Using the pulsepulse labeling method, we analyzed 110 different protein spots. In all but 2 of the 110 spots, new pulse counts divided by old pulse counts remained constant, with some minor variation through the cell cycle. However, the variation was much less than the twofold changes in ratio predicted by mechanisms other than exponential synthesis. This means that the apparent rate of synthesis increases exponentially during the cell cycle. Methionine was used for pulse labeling, so the apparent rate approximates the true rate. That is, pool corrections are negligible because the pool for methionine is half-saturated within 20 s (14). Furthermore, the specific activity of the methionine pool remains constant during the cell cycle (manuscript in preparation). The unusually high average ratios observed for proteins 24 and 209, 1.03 and 2.26, respectively, versus an
VOL. 137, 1979 13.
REGULATION OF PROTEINS DURING THE CELL CYCLE FLOW RATE (mllmin) 17 19 21 23
15
methylation,
.6
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or
significant molecular weight
change. Furthermore, noncovalent addition of
27
25
1189
regulatory molecules
or
regulatory proteins
to
protein complexes, or monomer-dimer changes, would not be detectable since the conditions of electrophoresis result in completely denatured proteins. This work confirms the results of a previous study which used a long-term label followed by a short pulse label (1). In that study, it was
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FIG. 4. Ratio ofpulse radioactivities of a protein through the cell cycle. Protein was dual-labeled and fractionated by elutriation. Electrophoresis was performed on each fraction. For each fraction, spot 67 (Fig. 3) was cut out and the ratio of radioactivites in it was determined. For explanation of the assignment of cell cycle position, see reference 1.
of 0.36 for all 110 spots, are indicative of either increased degradation or modification. These same two proteins were examined by the long-term/pulse dual-label method and neither showed any periodic variations in ratio (1). Thus, it is unlikely that the periodicity observed here is due to periodic synthesis or step synthesis. This suggests that the variations in these two proteins are probably due to periodic degradation or modification. The pulse-pulse labeling method described here can distinguish between various patterns of protein accumulation. Since the rate of synthesis through the cell cycle is computed from the ratio of two pulse labels separated in time, degradation and some types of protein modifications can be detected. If new and old proteins are modified or degraded differentially, then the 35S/3H ratio will change as if there was a sudden increase or decrease in the rate of synthesis of the protein. Any periodic modification that alters the electrophoretic mobility of the polypeptide in either dimension would also result in a change in ratio. Since, in general, no changes were observed, we suggest that neither periodic modification that alters the position of two-dimensional gels nor periodic degradation is a general characteristic of the yeast cell cycle. However, some types of modifications would not be detected by this method. These would include those modifications that do not result in a charge change, e.g., average
1.
'S/3H
ratios of individual proteins
through the cell cycle Flow rate
tein
(ml/min)a
17 13 15 19 21 23 25 27 1 0.12 0.13 0.13 0.09 0.12 0.10 0.10 0.12 3 0.43 0.39 0.42 0.30 0.31 0.35 0.30 0.32 7 0.70 0.82 0.69 0.61 0.75 0.81 0.79 0.90 9 0.27 0.28 0.27 0.28 0.29 0.20 0.26 0.26 10 0.47 0.33 0.43 0.34 0.30 0.32 0.33 0.26 12 0.13 0.12 0.13 0.15 0.13 0.17 0.14 0.11 24 2.15 1.23 1.63 0.96 0.60 0.21 0.64 0.78 36 0.27 0.31 0.30 0.26 0.32 0.25 0.23 0.29 39 0.62 0.65 0.66 0.62 0.72 0.74 0.74 0.75 43 0.19 0.18 0.16 0.16 0.16 0.15 0.16 0.14 45 0.34 0.29 0.29 0.29 0.28 0.22 0.27 0.27 47 0.28 0.23 0.25 0.23 0.31 0.23 0.24 0.24 61 0.10 0.13 0.12 0.12 0.10 0.12 0.11 0.12 63 0.11 0.12 0.10 0.12 0.10 0.13 0.13 0.06 67 0.27 0.26 0.23 0.23 0.24 0.25 0.23 0.22 68 0.53 0.46 0.41 0.47 0.51 0.38 0.52 0.34 79 0.22 0.21 0.20 0.21 0.20 0.21 0.19 0.18 85 0.25 0.25 0.24 0.24 0.22 0.22 0.21 0.20 88 0.64 0.55 0.57 0.61 0.58 0.58 0.61 0.60 144 0.22 0.22 0.17 0.19 0.23 0.31 0.19 0.18 146 0.23 0.25 0.26 0.26 0.27 0.26 0.26 0.26 161 0.39 0.27 0.35 0.32 0.29 0.40 0.32 0.32 163 0.40 0.39 0.43 0.35 0.40 0.42 0.36 0.27 167 0.29 0.31 0.38 0.35 0.35 0.21 0.26 0.27 169 0.18 0.22 0.16 0.18 0.19 0.19 0.16 0.18 171 0.19 0.16 0.17 0.13 0.24 0.21 0.15 0.15 182 0.26 0.28 0.23 0.25 0.20 0.35 0.31 0.22 183 0.19 0.17 0.18 0.17 0.17 0.17 0.16 0.15 184 0.24 0.24 0.22 0.26 0.24 0.26 0.21 0.22 186 0.29 0.23 0.23 0.23 0.27 0.22 0.22 0.25 193 0.21 0.20 0.19 0.21 0.17 0.22 0.20 0.20 200a 0.21 0.20 0.20 0.20 0.19 0.19 0.18 0.18 208 0.33 0.27 0.23 0.27 0.26 0.28 0.23 0.29 209 3.00 2.69 3.39 2.11 2.50 1.54 1.71 1.13 217 0.26 0.22 0.21 0.23 0.23 0.26 0.23 0.28 225 0.24 0.33 0.27 0.23 0.24 0.31 0.22 0.28 227 0.25 0.19 0.18 0.17 0.21 0.19 0.19 0.18 229 0.58 0.48 0.48 0.49 0.45 0.48 0.47 0.48 231 0.20 0.20 0.19 0.17 0.18 0.19 0.17 0.16 232 0.16 0.15 0.15 0.15 0.16 0.15 0.14 0.13 236 0.23 0.22 0.24 0.21 0.23 0.23 0.27 0.24 241 0.39 0.31 0.31 0.32 0.31 0.32 0.30 0.27 243 0.32 0.29 0.66 0.26 0.25 0.18 0.27 0.29 251 0.21 0.20 0.19 0.19 0.20 0.19 0.18 0.15 252 0.55 0.45 0.49 0.43 0.45 0.30 0.43 0.46 253 0.29 0.24 0.24 0.24 0.25 0.25 0.23 0.22 254 0.29 0.26 0.24 0.21 0.26 0.24 0.20 0.20 255 0.31 0.28 0.26 0.27 0.25 0.26 0.23 0.25 282 0.43 0.38 0.34 0.33 0.26 0.44 0.34 0.45 287 0.34 0.41 0.26 0.29 0.28 0.29 0.29 0.29 a The relationship between flow rate and cell cycle position can be deduced from Fig. 4.
J. BACTERIOL. 1190 ELLIOTT AND McLAUGHLIN shown that 111 different protein spots have an modification through the cell cycle compels a exponentially increasing rate of synthesis during rethinking of the present models controlling cell the cell cycle. Including the 110 proteins ana- division. lyzed here, 130 different polypeptide spots, numACKNOWLEDGMENTS bered in Fig. 3, were analyzed, and all showed were supported by Public Health SerThese investigations of the rate of Ninety an exponential synthesis. CA10628 awarded by the National Cancer Institute. 130 were tested by both the long-term label- viceWegrant thank Riley and Anita Newman for helpful discussions. pulse (1) and the pulse-pulse method. Since these 130 were selected randomly from the over LITERATURE CITED 550 spots that are detectable on autoradiograns S. and C. S. McLaughlin. 1978. Rate of G., 1. Elliott, of two-dimensional gels, we suggest that periodic macromolecular synthesis through the cell cycle of the variation in the rate of synthesis is not a general yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. characteristic of the cell cycle. U.S.A. 75:4384-4388. of Clearly, our results establish that for most 2. Gordon, C. N., and S. G. Elliott 1977. Fractionation Saccharomyces cerevisiae cell populations by centrifuproteins there is little or no periodic modificagal elutriation. J. Bacteriol. 129:97-100. tion, degradation, or change in the rate of syn- 3. Halvorson, H. O., B. L A. Carter, and P. Tauro. 1971. thesis during the cell cycle. However, our system Synthesis of enzymes during the cell cycle. Adv. Microb. Physiol. 6:47-106. does not resolve the basic and acidic proteins, L H. 1967. Macromolecule synthesis in temand the less abundant class of proteins was not 4. Hartwell, perature-sensitive mutants of yeast. J. Bacteriol. 93: analyzed. Regulatory proteins may exhibit pe1662-1670. riodic behavior since they are present often in 5. Hartwell, L H. 1970. Periodic density fluctuations during the yeast cell cycle and the selection of synchronous amounts of only a few copies per cell. In fact, cultures. J. Bacteriol. 104:1280-1285. periodic synthesis has been demonstrated for 6. Hartwell, L H., J. Culotti, J. Pringle, and B. Reid. in our system (9). histones, which do not run 1974. Genetic control of the cell division cycle in yeast. Whether our results can accommodate the nuScience 183:46-51. merous observations on the periodic nature of 7. Mitchison, J. M. 1969. Enzyme synthesis in synchronous cultures. Science 166:657-663. enzyme activity during the cell cycle (3, 7) is J. M. 1971. The biology of the cell cycle. uncertain. Many of these observations might in 8. Mitchison, University Press, Cambridge, U.K. fact be due to variations in enzyme activity. 9. Moll, R., and E. Wintersberger. 1976. Synthesis of yeast histones in the cell cycle. Proc. Natl. Acad. Sci. U.S.A. A number of general theories on the nature of 73:1863-1867. control of periodic synthesis, such as gene dou- 10. O'Farrell, P. H. 1975. High resolution two-dimensional bling, oscillatory repression, and linear reading, electrophoresis of proteins. J. Biol. Chem. 250:4007have been proposed to account for the observa4021. tions on the periodic nature of proteins. How- 11. Sanderson, R. J., and K. E. Bird. 1977. Cell separations by counterflow centrifugation. Methods Cell Biol. 15:1ever, these models appear unlikely for the ma14. jority of the proteins in S. cerevisiae. Doubling 12. Sanderson, R. I., K. E. Bird, N. F. Palmer, and J. in DNA has been shown to have no effect on the Brenman. 1976. Design principles for a counterflow centrifugation cell separation chamber. Anal. Biochem. rate of synthesis of other molecules, including 71:615-622. total protein, total RNA, double-stranded RNA, 13. Sebastian, J., B. L A. Carter, and H. 0. Halvorson. rRNA, tRNA, polyadenylic acid-containing 1971. Use of yeast populations fractionated by zonal RNA, and ribosomal proteins (1; manuscripts in centrifugation to study the cell cycle. J. Bacteriol. 108: 1045-1050. preparation). Therefore, it appears that the cell J. R., S. A. Morgan, and R. W. Shumnan. does not immediately use the additional DNA 14. Warner, 1976. Kinetics of labeling of the S-adenosylmethionine made during S phase. Continuous synthesis of pool of Saccharomyces cerevisiae. J. Bacteriol. 125: proteins with no periodic degradation and/or 887-891.
