Vol. 11, No. 7

MOLECULAR AND CELLULAR BIOLOGY, JUlY 1991, p. 3691-3698, 0270-7306/91/073691-08$02.00/0 Copyright © 1991, American Society for Microbiology

Protein Synthesis Requirements for Nuclear Division, Cytokinesis, and Cell Separation in Saccharomyces cerevisiae DANIEL J. BURKE* AND DEANNA CHURCHt

Department

of Biology, Gilmer Hall, University of Virginia, Charlottesville, Virginia 22901

Received 19 February 1991/Accepted 25 April 1991

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (WC), and cdc2l (also named tmpl) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36C), cdc2O, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.

The cell cycle of the yeast Saccharomyces cerevisiae has been extensively analyzed from a genetic perspective. There are over 50 genes required for completing the cell division cycle defining the CDC genes (31). cdc mutants have been used to determine the organization of stage-specific events within the cell cycle (13, 31) and to test whether stagespecific events in the cell cycle result from stage-specific expression of CDC gene products. Analysis of single and double mutants and reciprocal-shift experiments suggest that temporal order is achieved by sequential action of genes such that late events are not executed until earlier events are completed (14, 17, 25, 31, 45). The cell cycle is dependent on the order of function of CDC gene products and independent of their order of synthesis. Other genetic experiments suggest that most CDC gene products are present in a large excess, sufficient to support several cell divisions (2). Additional supporting evidence comes from two-dimensional polyacrylamide gel electrophoresis of newly synthesized proteins in wild-type cells. Most proteins are synthesized constitutively, and there is little periodic protein synthesis during the cell cycle (6, 20). These experiments, taken together, suggest that order of function and not order of synthesis of proteins is a primary determinant of temporal order during cell division. In this way, the cell cycle in S. cerevisiae resembles bacteriophage morphogenesis (46). During phage assembly, most gene products are synthesized in excess and temporal control of phage assembly is primarily regulated by the order of function and not the order of synthesis of phage proteins. The analysis of S. cerevisiae cell division described above addresses only the requirement to synthesize the majority of proteins and does not rule out the possibility that there is temporally regulated protein synthesis of a small number of important polypeptides. There is good evidence, from a variety of experimental systems, that protein synthesis is required to complete some regulatory steps in the cell cycle. Initiating DNA synthesis and initiating mitosis are particularly sensitive to protein synthesis inhibitors. There is a need

for rapid protein synthesis during G1, in proliferating mammalian fibroblasts, to synthesize an important regulatory protein (30). There is also a requirement for protein synthesis, in a variety of organisms, to complete the G2 phase and enter mitosis (5, 7, 9, 38, 42). Recent experiments provide a molecular explanation for the mitotic regulation in terms of a ubiquitous regulatory factor termed mitosis-promoting factor (MPF). MPF is regulated by proteins called cyclins, and both MPF and cyclins are present in organisms ranging from yeasts to humans (7, 9, 10, 38, 42). Biochemical evidence has shown that MPF is activated when cyclin proteins accumulate above a threshold (23, 28, 29). The earlier observation that entering mitosis requires protein synthesis can be explained, in part, by the requirement to accumulate cyclin (23, 28). Protein synthesis inhibitors have been used to study the requirement for protein synthesis during the cell cycle of S. cerevisiae; however, experiments have focused primarily on the G1 phase of the cycle. Coordinating growth and cell division occurs in G1 at a regulatory step called START. Under favorable growth conditions, which include sufficient protein synthesis, cells will initiate a cell cycle. Limiting protein synthesis extends the length of the G1 phase but has little effect on the timing of other events (19, 39). These observations are interpreted in terms of a size requirement for cell cycle initiation (19, 31) that may depend upon the accumulation of a labile protein (36). The temporal requirement for protein synthesis in the G1 phase was mapped more precisely relative to the a-factor-sensitive step and cdc mutants. The data suggest that there is a second requirement for protein synthesis, in addition to START, to complete G1 and initiate DNA synthesis. Cells previously arrested with a-factor do not initiate DNA synthesis in the absence of protein synthesis (16, 43), and cdc25 mutants delay bud initiation in response to pulses of cycloheximide. cdc4 mutants arrested at the restrictive temperature do not initiate DNA synthesis when returned to the permissive temperature in the presence of cycloheximide (17). Cells aligned at the CDC7-dependent step complete DNA synthesis in the presence of cycloheximide (16). The requirement for protein synthesis for completing the G1 phase of the cell cycle and initiating DNA synthesis is after the CDC4-dependent step and before the CDC7-dependent step. Temporal mapping

Corresponding author. t Present address: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139. *

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BURKE AND CHURCH

MOL. CELL. BIOL.

TABLE 1. Yeast strains

Straina

Relevant genotype

700-3-3 H2C2A2 H6C1A1 H7C1A5 H8C1A1 H9C1A1 H13C1A1 H14C1A1 H15C2A1 H16C1A2

Wild type cdc2-2 cdc6-1 cdc7-5 cdc8-1 cdc9-1 cdc13-1 cdc14-1 cdcis-I cdc16-2

H17C1A1 H20C1A1 H23C1A1 RS167

cdcl7-1 cdc20-1 cdc23-1 tmpl-6 tutl-2 (pJM81)

Source

This study L. Hartwell L. Hartwell L. Hartwell L. Hartwell L. Hartwell L. Hartwell L. Hartwell L. Hartwell L. Hartwell L. Hartwell L. Hartwell L. Hartwell R. Sclafani

a All strains are MATa. All strains are isogenic his7 ural mutants congenic with A364A (15), except for RS167, a nonisogenic auxotrophic mutant congenic with A364A.

using cdc mutants suggest that there may be two requirements for protein synthesis in Gl: one for completing START and the other, after the CDC4-dependent step, for initiating DNA synthesis. S. cerevisiae cells accumulate in G, under starvation conditions, when nutrients are limited (19, 31, 39). Cells that are starved for nitrogen after passing START complete a cell cycle with less than a 10% increase in cellular protein (19). If the proteins necessary to complete the stage-specific events in the cell cycle are in excess and present at the completion of START, then cells should be able to complete a cell cycle when protein synthesis is completely inhibited. However, treating cells with cycloheximide, at sufficient concentrations to inhibit protein synthesis, arrests cell division and cells accumulate at multiple points in the cell cycle (31). Therefore, the small amount of protein synthesis (10%) that occurs under nitrogen starvation is essential to completing cell division. Protein synthesis must be required for completing events in the cell cycle after START. We have used two protein synthesis inhibitors (cycloheximide and trichodermin) and cdc mutants to map the temporal requirements for protein synthesis after the G, phase of the cell cycle in S. cerevisiae. The ability to complete landmark events (15) was determined by a combination of techniques including flow cytometry, fluorescence microscopy, and phase-contrast microscopy. We show that there is a protein synthesis requirement at two points in the cell cycle after the G, phase. Protein synthesis is required to progress from the end of DNA synthesis to nuclear division and to complete cytokinesis and cell separation. MATERIALS AND METHODS Growth and maintenance of strains. Strains and relevant genotypes are listed in Table 1. Routine maintenance of yeast strains was as described previously (35). All strains were grown in YEPD plus 2% agar or in liquid YM-1 (11) containing 2% glucose. Strain RS167 was supplemented for growth with 100 ,ug of thymidine (Sigma) per ml (34). SC-leucine medium (35) was used for determining incorporation of radioactively labelled leucine to measure protein synthesis. Cycloheximide (Sigma) and trichodermin (gift from Leo Pharmaceuticals, Denmark), used at 10 p.g/ml, were added directly to the medium. Cells were synchronized

by centrifugal elutriation as described (4). Nocodazole (Sigma) was used at 15 ,ug/ml as described previously (18). Protein synthesis. The rate of protein synthesis was measured after overnight growth in SC medium lacking leucine. Cells were grown in 5-ml cultures of SC containing 10 ,ug of leucine per ml and 5 ,uCi of L-[4,5-3H(N)] leucine (NEN Dupont) per ml. We measured incorporation of radioactive leucine into protein as described earlier (37). Microscopy. Cells were sonicated by five bursts (20 W each) of a Branson 250 sonifier before examination. Cells were analyzed for bud morphology by phase-contrast microscopy using a Zeiss Universal compound microscope. Nuclear division was assayed by fluorescence microscopy using a Zeiss axiophot photomicroscope, after being stained with 0.5 ,ug of 4',6-diamidino-2-phenylindole (DAPI) (Sigma) (44). Cells were analyzed by flow cytometry as described earlier (1). Experimental design. Strains were grown at 23°C to an approximate cell density of 106 per ml. Cells were sonicated and examined by phase-contrast microscopy, and the total number was determined by counting with the aid of a hemacytometer. An aliquot was removed, diluted, and plated onto YEPD plates to determine the plating efficiency. The culture was then grown at 36°C for a period determined as the time required for at least 85% of the cells to acquire the terminal budded phenotype (31). Cells were sonicated, and a sample was removed, diluted, and placed onto YEPD plates to determine the viability after incubation at 36°C. The culture was divided in half; cycloheximide was added to one half, and the other half was untreated. Cultures were returned to 23°C after 2 min and a-factor (Sigma) was added to 10-7 M. Cultures were maintained at 23°C, and samples were removed at various times. Cells that were analyzed by DAPI staining were fixed overnight in 3.7% formaldehyde. Cells prepared for flow cytometry were fixed for 1 h in 75% ethanol before overnight staining in propidium iodide (1). The above procedure resulted in cells that retained at least 50% viability after incubation at 36°C for all cdc mutants except cdc7. Prolonged incubation of strain H7C1A1 (cdc7) at 36°C resulted in high inviability. In order to shorten the incubation time at the restrictive temperature, we obtained a synchronous population of viable cdc7 cells by centrifugal elutriation (4) after growth at 23°C. We incubated the cells for 45 min at the restrictive temperature to arrest the cells at the CDC7-dependent step. Mutants that arrest in S phase after incubation at the restrictive temperature (cdc2, cdc6, cdc8, cdcl7 [38°C], and cdc2l) have terminal phenotypes by DAPI staining similar to those of the mutants that arrest in G2 (cdc13, cdc16, cdc17 [34°C], cdc2O, and cdc23). The S-phase mutants and the G2 mutants arrest as large budded cells with a single DAPIstaining nucleus (31). We scored the ability to complete nuclear division by the number of DAPI-stained nuclei per cell. cdc mutants that arrested as budded cells with a single DAPI-stained nucleus (medial nuclear division) were analyzed for the appearance of binucleate cells (late nuclear division) or completion of cell separation and arresting as unbudded cells in the presence of a-factor. Cells from cdc mutants that were budded and retained a single DAPIstained nucleus were scored as being unable to complete nuclear division. RESULTS Protein synthesis inhibitors arrest growth and cell division. The strains listed in Table 1 are sensitive to both cyclohex-

NUCLEAR DIVISION AND CELL SEPARATION IN S. CEREVISIAE

VOL. 11, 1991

3693

TABLE 2. Effect of inhibitors on cell and nuclear morphologies Treatment

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imide and trichodermin, as growth ceased immediately after the drugs were added. We determined the extent of protein synthesis inhibition by measuring the rate of protein synthesis, before and after the addition of drugs, in strain 700-3-3, as described in Materials and Methods. Cycloheximide acted immediately and inhibited the rate of protein synthesis by at least 95% (data not shown). Similar results were obtained with trichodermin and the wild-type strain 700-3-3. There were similar effects on growth and protein synthesis for all strains used in the experiments (data not shown). The effect of inhibiting protein synthesis on the distribution of cell morphologies in the wild-type strain 700-3-3 was assayed by phase-contrast microscopy (budding) and DAPI fluorescent staining (nuclear division). We treated a random population of dividing cells with 10 p.g of cycloheximide or trichodermin per ml for 2 h and then fixed and stained the cells with the DNA-specific dye DAPI. The proportions of cells with different budded morphologies and with different nuclear morphologies were similar after treatment with cycloheximide (Table 2). cdc mutants that arrest in S phase complete DNA replication but not nuclear division without protein synthesis. Earlier studies showed that DNA synthesis, once initiated, could be completed in the presence of cycloheximide (16, 43). We used flow cytometry to assay DNA content and to determine whether cdc mutants that were arrested in S phase could complete DNA replication when protein synthesis was inhibited. Figure 1 shows the response of the wild-type strain 700-3-3 to cycloheximide. The data show relative DNA contents in a population of cells as measured by propidium iodide fluorescence. In asynchronous wild-type cells (Fig. 1A) there are characteristic subpopulation peaks of G1 and G2 cells, separated by cells that have initiated DNA synthesis and contain an intermediate amount of DNA. After cycloheximide treatment (Fig. 1B), only the G1 and G2 peaks remain. These data show that propidium iodide staining and flow cytometry can be used to monitor the effects of cycloheximide on DNA synthesis in a population of cells. Our results, in agreement with previous results, provide a simple way to show that cycloheximide does not inhibit DNA replication (16, 43). cdc7 mutants arrest early in S phase after completing a limited amount of DNA synthesis (21). Hereford and Hartwell (17) used incorporation of radioactive precursors into DNA to show that cells arrested at the CDC7 step complete DNA synthesis when incubated under permissive conditions in the presence of cycloheximide. We have repeated these experiments using flow cytometry and extended them to other DNA synthesis mutants. Cells from strain H7C1A2 (cdc7) were grown at 36°C to align the cells at the CDC7dependent step. After returning the cells to 23°C, we added cycloheximide and prepared cells, at various times, for flow cytometry. The data (Fig. 1C) show that the cells aligned at the cdc7-dependent step have a G1 content of DNA (in) and, after cycloheximide treatment (Fig. 1D), complete the bulk

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FIG. 1. Flow cytometry to measure the extent of DNA synthesis in response to cycloheximide. Wild-type strain 700-3-3 was grown at 23°C in YM-1, and asynchronous cells were prepared for flow cytometry (panel A). The same cells were treated for 1 h, in YM-1 containing 10 ,ug of cycloheximide per ml (panel B). cdc7 cells were isolated after growth at 23°C followed by centrifugal elutriation to obtain G1 cells and then incubated at 36°C for 45 min (panel C). The cdc7 cells were returned to 23°C, cycloheximide was added to 10 ,ug/ml, and the cells were incubated for 60 min (panel D).

of DNA synthesis and accumulate with a G2 content of DNA (2n). We repeated this experiment for four other strains that arrest in DNA synthesis, H2C1A1 (cdc2), H6C1A1 (cdc6), H8C1A1 (cdc8), and RS167 (cdc2l, also named tmpl) and obtained similar results (not shown). Each mutant cdc2, cdc6, cdc8, and cdc2l arrested as a heterogenous population in S phase but completed DNA synthesis in the presence of cycloheximide. We used DAPI staining to determine whether the cdc mutants that completed DNA synthesis in the presence of cycloheximide could complete nuclear division. Cells were aligned in S phase by incubating under restrictive conditions, and half of the cells were treated with cycloheximide. We allowed the cells to recover from the cdc block under permissive conditions, and the proportion of cells with divided nuclei was determined by staining with DAPI. The data for four mutants, cdc2, cdc6, cdc7, and cdc8, are shown in Fig. 2. The temperature-sensitive mutants were partially inviable after incubating at the restrictive temperature (indicated by **). We assume that this explains why only approximately half of the control cells (without cycloheximide treatment) can recover from the restrictive condition and complete nuclear division in each experiment. The kinetics of recovery from the restrictive condition and completing nuclear division differed for each strain. None of the mutants could complete nuclear division in the presence of cycloheximide. We repeated the experiments with strains H6C1A1 (cdc6) and H8C1A1 (cdc8) and trichodermin with the same results (not shown). Therefore, the inability to complete nuclear division in the presence of cycloheximide was due to inhibited protein synthesis. In both strains, cells did not complete nuclear division at the permissive temperature when recovering from the restrictive condition in the presence of trichodermin (not shown).

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FIG. 3. Ability of mutants arrested in G2 to complete nuclear division in cycloheximide. Mutants (panels B to D) were aligned at the CDC steps by incubating at the restrictive temperature. Wildtype cells (panel A) were treated with nocodazole as described in Materials and Methods. Cells were stained with DAPI, and the percentage that remained arrested was determined. Cells were returned to permissive conditions in the presence (0) or absence (0) of cycloheximide. (A) Nocodazole treated wild type. (B) H20C1A1 (cdc2O). (C) H23C1A1 (cdc23). (D) H16C1A2 (cdcl6).

We also determined the viability of cells that completed DNA replication when protein synthesis was inhibited. At least 40% of the cells could form colonies after incubation in cycloheximide. Since approximately 50% of the cells in each experiment were inviable after each experiment (see above), we conclude that most cells that complete DNA synthesis in the presence of cycloheximide can complete cell division after the inhibitor is removed. DNA synthesized in the presence of cycloheximide is a suitable substrate for mitosis. cdc mutants aligned in the G2 phase complete nuclear division without protein synthesis. We tested several cdc mutants that arrest in G2 for their ability to complete nuclear division in the presence of cycloheximide. Wild-type cells arrest in G2 when treated with nocodazole, a microtubule inhibitor (18). Cells were aligned in the G2 stage of the cell cycle by incubation under restrictive conditions and then returned to permissive conditions in the presence or absence of cycloheximide. We stained cells with DAPI to determine the fraction that could complete nuclear division. The data are shown graphically in Fig. 3. In each case, the cells previously arrested in G2 were able to complete nuclear division under permissive conditions without concomitant protein synthesis. The kinetics differed for each mutant. For example, the data for cdc23 mutants (Fig. 3C) are on a different time scale than other mutants because cells recovering from the restrictive temperature completed nuclear division slowly. In contrast, cdc2O mutants (Fig. 3B) completed nuclear division quickly. We tested the effect of a different inhibitor of protein synthesis on two of the strains. We obtained similar results with strain H2OC1A1 (cdc2O) and strain 700-3-3 (nocodazole treated) in the presence of trichodermin.

To determine the viability of the cells that completed mitosis in the presence of cycloheximide, we washed the cells free of the inhibitor and plated them at 23°C. In each case, at least 75% of the cells were viable and could form colonies after cycloheximide treatment. We conclude that cells arrested in G2 complete nuclear division successfully in the presence of cycloheximide. Cells complete nuclear division from the RAD9 step without protein synthesis. The RAD9 gene controls the cellular response to DNA damage by causing cells to arrest in the G2 stage of the cell cycle at a point where the DNA damage can be repaired (41). Several cdc mutants arrest in the G2 stage in a RAD9-independent manner. Other cdc mutants arrest in G2 only if the RAD9 gene is functional, suggesting that the mutants accumulate DNA damage and the cell cycle arrests as a consequence of stopping to repair the DNA. For example, the CDC17 gene encodes DNA polymerase I (3) and cdc17 mutants arrest in G2 when incubated at 34°C and arrest in S phase when incubated at 38°C (3). Arresting cdcl7 cells in G2 is dependent on the function of the RAD9 gene (41). The interpretation is that 38TC is completely restrictive for cdcl 7 mutants and cells arrest during DNA synthesis. At 34°C, the CDC17 gene product is partially functional and completes DNA replication with low fidelity. The RAD9 function therefore arrest cells in G2 to permit the damaged DNA that accumulates at 34°C to be repaired. We tested the ability of cdc17 mutants to complete nuclear division in the presence of cycloheximide after incubation at either 38°C (arrested in S phase) or 34°C (arrested in G2). The data (Fig. 4) show that cdcl7 cells arrested at 34°C (Fig. 4A) at the RAD9-dependent step behave like the other mutants that arrest in G2. They complete nuclear division in the presence

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NUCLEAR DIVISION AND CELL SEPARATION IN S. CEREVISIAE

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Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Sacc...
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