Characterization of the fission yeast cdc10 + commitment to the cell cycle. protein that is required for. VIESTURS SIMANIS and PAUL NURSE.

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1 Characterization of the fission yeast cdc0 + commitment to the cell cycle protein that is required for VIESTURS SIMANIS and PAUL NURSE Imperial Cancer Research Fund, Cell Cycle Control Laboratory, PO Box 23, Lincoln's Inn Fields, London \YC2A, 3PX, UK and Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford 0X 3QU, UK Summary We have used antiserum raised against a /J-galactosidase-cdcKT " fusion protein to identify the protein product of the cdclo+ start gene of Schizosaccharomyces pombe. This gene is required for progress through the Gi phase of the cell cycle and for activating processes such as the increase in histone mrna level in preparation for S phase. The protein has an apparent molecular weight of and is phosphorylated on multiple serine residues. The protein remains phosphorylated throughout the mitotic cell cycle and shows no significant steady-state changes in level. The antiserum has also detected a protein similar in size to pst 0 * 00 in human cells. Key words: cell cycle, Schizosacchammyces phosphoprotein. pombe, G, Introduction One of the control points in the cell cycle of the fission yeast Schizosacchammyces pombe is known as 'start'. This is the point in Gi where cells become committed to the mitotic cell cycle with respect to the alternative developmental fate of conjugation and initiate the processes leading to S phase (Fantes, 984; Matsumoto et al. 987). Start may be considered to divide the yeast cell cycle functionally into two parts. An uncommitted phase during the first part of Gi, and a committed phase comprising the rest of Gi, S phase, G 2 and mitosis. A start control also operates in budding yeast (Pringle & Hartwell, 98; Reed, 984) and a similar control point has also been identified in mammalian cells (Pardee et al. 978). A large collection of temperature-sensitive mutants blocked in cell cycle progress has been generated (Nurse et al. 97; Nasmyth & Nurse, 98). Representatives of these mutants have been screened for their ability to conjugate when arrested at the restrictive temperature (Nurse & Bissett, 98). Mutants in two genes, cdc2 + and cdclo +, were able to conjugate and were thereby operationally defined as being arrested in Gi before the traverse of start. During rapid growth and Gi phase of the cycle is very short, so the cells pass start early in the cell cycle, soon after mitosis. In cell size mutants (Nurse, 975) and at slow growth rates (Nasmyth, 979) Gi becomes expanded. However, the time from the execution of cdclo + gene function to the onset of mitosis is essentially invariant, irrespective of growth rate (Nasmyth,' 979), Journal of Cell Science 92, 5-5 (989) Printed in Great Britain The Company of Biologists Limited 989 indicating that at slow growth rates start is traversed much later in the cell cycle. Measurements of cell size at S-phase (Nurse & Thuriaux, 977) have suggested that a minimum cell mass must be obtained before a cell can undergo S phase. Taken together these data establish that at slow growth rates traverse of start and execution of cdclo + gene function is rate-limiting for entry into the mitotic cell cycle. The cdclo + gene transcript does not vary significantly in level during either entry into stationary phase or passage through the mitotic cell cycle (Aves et al. 985), so the execution of cdclo + gene function leading to commitment to the cell cycle is not regulated at the level of transcription. Sequencing of the cdc!0 + gene has identified a single long open reading frame with the potential to encode a protein of M r (Aves et al. 985). The putative protein has homology with theswi gene product of the budding yeast, which is involved in control of HO gene transcription and thereby the regulation of mating type switching (Breeden & Nasmyth, 987). In this paper we report the production of antisera that identify the protein encoded by the cdclo + gene. We demonstrate that the gene encodes an A/ r protein phosphorylated on multiple serine residues. We have also studied the behaviour of the protein through the mitotic cell cycle and upon entry into stationary phase. Materials and methods Strains S. pombe strains 972h~ iueel-s0h~ and Ieul-32h~ have been 5

2 described (Nurse, 975; Beach & Nurse, 98). Bacterial strain DH5 (Hanahan, 983) was used for routine cloning procedures and strain BMH 7-8 (Ruther & Muller-Hill, 983) was used for production of fusion protein. S. pombe and Escherichia coli were transformed according to standard procedures (Beach & Nurse, 98; Hanahan, 983). Plasmids The plasmids pur292 (Ruther & Muller-Hill, 983), pevpll (Russell & Nurse, 98) and pcdcl0.2 (Aves et al. 985) have been described. All other plasmids were constructed as described below using standard techniques (Maniatis et al. 982). Plasmid ptb2 consists of the 2- kb (03 bases) Sstl-Hindlll fragment of pcdcl0.2 (Aves et al. 985) cloned into the corresponding sites of pevpll. In this plasmid the cdcj0+ gene is transcribed from the S. pombe alcohol dehydrogenase promoter and was used to overexpress the gene product. Plasmid pfp3 consists of the l-4kb Pstl-Hindlll fragment encoding the C-terminal 329 amino acids of the cdclo+ gene ligated to the corresponding sites of pur292, thus generating a cdclo+-figalactosidase fusion protein, which was used to raise antisera to identify the cdci0+ protein. Antibody production Cultures of BMH 7-8 bearing pfp3 were grown under selective conditions to late log phase and induced to produce fusion protein by addition of IPTG to mm. Cells were harvested by centrifugation and fusion protein was purified by preparative electrophoresis on 5 % (w/v) polyacrylamide gels (Laemlli, 970). The fusion protein was electroeluted from the gel, precipitated with acetone and resuspended in phosphatebuffered saline (PBS). Rabbits were injected with approximately 200 ^(g of protein emulsified in complete Freund's adjuvant at multiple subcutaneous sites. Four weeks later, and at intervals thereafter, booster injections of about 200 fig of protein emulsified in incomplete Freund's adjuvant were given and the animals were bled 7-0 days later. Serum was depleted of antibody to /Sgalactosidase by passage through a column of /S-galactosidase (Sigma) linked to Reactigel (Pierce). Antibody to cdcl0+ protein was then purified by affinity chromatography on a column of /S-galactosidase cdcl0+ fusion protein. A 5 ml sample of serum was incubated overnight with 3 ml of Reactigel resin to which approximately 5 ml of /J-galactosidase had been coupled. The unbound material was collected and applied to ml of Reactigel resin to which approximately 3 mg of fusion protein had been coupled. After overnight incubation the resin was poured into a column, the flowthrough collected, and the column washed extensively with PBS (0vol.), PBS + M-KC (20 vol.) and PBS (5 vol.). The bound antibody was eluted with 0' M-glycine HC, ph 2, and neutralized with 0-5M-sodium phosphate buffer, ph7-5. The antibody was adjusted to 30% (v/v) glycerol and stored at 70 C. All assays were performed in antibody excess. Cell labelling and protein analysis Conditions for growth and labelling of cells, immunoprecipitations, one- and two-dimensional electrophoresis and Western blotting have been described (Simanis & Nurse, 98). Synchronous cultures were generated using a Beckman JE0X elutriator rotor (Aveset al. 985). Cell number was determined using a model ZM Coulter Counter and cell length was measured using a Zeiss drum micrometer eyepiece as described by Russell & Nurse (987). Phosphoamino acid analysis and phosphopeptide mapping were performed according to Hunter & Sefton (980) and Beemon & Hunter (978). 52 V. Simanis and P. Nurse Results Identification o/cdcl0 + gene product To prepare an antibody to identify the cdcl0+ gene product, a fusion protein between cdcl0+ and /J-galactosidase was produced and used to immunize a number of rabbits. The resulting sera were first depleted of antibodies to /J-galactosidase, and then antibodies to the cdcl0+ part of the protein purified by affinity chromatography on solid-phase fusion protein. These antisera were used to probe Western blots of proteins extracted from S. pombe cells transformed with either pevpll or ptb2, which overexpresses the cdcl0+ gene. (For details of plasmid constructions, see Materials and methods.) The antibody identifies a protein of apparent molecular weight , which is present at much higher levels in cells transformed with ptb2 (Fig., lanes and 2). Labelling of cells with [ S]sulphate followed by immunoprecipitation identified a protein of similar molecular weight, , only when antibody to fusion protein was used but not when preimmune serum from the same animal was used in the reaction (Fig., lanes 3 and 4). These data establish that the protein product of the cdcl0+ gene is an 87000M r protein, hereafter referred to as p87. The observed molecular weight is in good agreement with that predicted from the DNA sequence, (Aves et al. 985). To determine if p87 cdcl was a phosphoprotein, cells transformed with either pevpll or ptb2 were labelled with [32P]phosphate and p87 cdcl immunoprecipitated Fig.. Characterization of antisera to the cdcw+ gene product. Lanes, 2: strain leu -32 was transformed with either pevpll (lane ) or ptb2 (lane 2) and protein extracts were prepared. After electrophoresis the proteins were transferred to nitrocellulose and incubated with antibody to gycdcio After washing, bound antibody was detected using ^I-labelled protein A followed by autoradiography. Lanes 3, 4: cells were labelled with [35S]sulphate and protein extracts immunoprecipitated either with preimmune serum (lane 3) or antibody to p87 cdcl protein (lane 4). Lanes 5, : cells transformed with either pevpll (lane 5) or ptb2 (lane ) were labelled with [32P]phosphate and protein extracts incubated with antibody to p87 cdcl protein. After washing the immunoprecipitates were analysed by gel electrophoresis. In lane, the nature of the additional 0K band is unknown, but it could be a proteolytic breakdown product of p87 cdcl.

3 In cells expressing wild-type levels of p87 cdcl a phosphorylated 87000M r protein was observed. In cells overproducing p87 cdcl a greater amount of this protein was observed (Fig., lanes 5 and ). These data establish that the product of the cdclo+ gene is a phosphoprotein of apparent molecular weight It was also noticed during the course of this study that overexpression of p87 cdcl affected cell size at division. Cells were found to be -3 times longer at division than wild-type when overexpressing p87 (9-4 ± 2- /im for ptb2 transformed cells compared with [im for pevpll transformed cells). The generation time of the cells was also longer than wild-type (225 min compared with 80min at 30 C in minimal medium). The finding that p87 cdcl was a phosphoprotein led us to try to identify the phosphorylated amino acid and the number of sites of phosphorylation. Cells were labelled with [32P]phosphate and p87 cdc0 immunoprecipitated. The protein was then eluted from the gel band, digested with trypsin and subjected to acid hydrolysis. Phospho- amino acid analysis by high-voltage thin-layer chromatography showed that only phosphoserine was present in the p87 cdcl protein (Fig. 2A). Two-dimensional peptide mapping following digestion with trypsin identified at least five phosphopeptides (Fig. 2B), indicating that pgycdcio j g phosphorylated at multiple sites. Two-dimensional gel electrophoresis followed by Western blotting identified a number of spots for p87 cdcl at an apparent isoelectric point of (Fig. 2C). These data are also consistent with the presence of multiple sites of phosphorylation within the p87 cdcl protein. Analysis of>#7cdcl through the mitotic cell cycle Earlier studies on the transcriptional control of the cdclo+ gene (Aves et al. 985) have shown that the steady-state transcript level of the gene does not vary significantly through the mitotic cell cycle. We have investigated whether there are changes in the protein level or phosphorylation of the protein during the cell cycle. A synchronous culture of weel-50 cells was pre- #.' T B i vi I >I Fig. 2. Phosphoamino acid analysis peptide mapping and two-dimensional gel electrophoresis of p87 cdcl. A. Phosphoamino acid analysis of p87 cdcl. Cells transformed with ptb2 were labelled with [ s P]phosphate and p87 cdcl immunoprecipitated. After digestion with trypsin the protein was hydrolysed with acid and the residue subjected to electrophoresis at ph 3-5. The positions of unlabelled phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) markers visualized by staining with ninhydrin are indicated. B. Phosphopeptide map of p87 cdc0 : part of the tryptic digest generated in A (above) was subjected to electrophoresis at ph -9 followed by ascending chromatography in acetic acid/88% formic acid/water (5:50: 794, by vol.). The directions of electrophoresis (E) and chromatography (C) are indicated. The circle represents the origin. C. Protein extracted from cells transformed with ptb2 was subjected to isoelectric focussing followed by SDS-polyacrylamide gel electrophoresis. The gel was Western blotted and probed with antibody to p87c'dclo as described in the legend to Fig.. The region of the gel shown spans the ph range ---, left to right. S. pombe cdclo pmtein 53

4 pared using an elutriator rotor and the culture was shifted to the restrictive temperature. In these conditions the mutant cells divide at a reduced size compared with wild type, leading to an expansion of the Gi phase before start (Nurse, 975; Russell & Nurse, 987). Use of this mutant should facilitate detection of any changes that occur in the Gi period before start, which is normally very brief in rapidly growing wild-type cells. The result of one synchronous culture is shown in Fig. 3. Fig. 3A demonstrates that good cell synchrony was achieved with two stepwise increases in cell number. A Western blot of protein extracts prepared from cells at various times throughout the cell cycle showed no significant variation in the amount of p87 cdcl (Fig. 3B). When cells were labelled with [32P] phosphate and immunoprecipitates prepared throughout the cell cycle it was found that the pgycdcio p r o t e j n remains phosphorylated during the whole of the mitotic cycle (Fig. 3C). These data indicate that the execution of cdcw+ gene function during the traverse of start is not regulated by modulation of p87 cdcl protein level or by extensive changes in the phosphorylation state of the protein. Analysts of p8tdcw during exit from the cell cycle Aves et al. (985) demonstrated that the cdclo* transcript level is not modulated during exit from the mitotic cycle. We have therefore investigated the level of p87 cdcl and its phosphorylation state as cells G\ arrest following nitrogen deprivation. Fig. 4A shows a Western blot of protein extracts prepared at various times after shifting wild-type cells to a nitrogen-deficient medium. It is clear that the level of the protein does not change appreciably as cells cease to proliferate. Fig. 4B shows immunoprecipitates prepared at similar times to those in Fig. 4A from cells labelled with [ 32 P]phosphate. This demonstrates that p87 cdcl remains phosphorylated as cells exit from the mitotic cycle, although small changes in phosphorylation level up to 2-5-fold estimated by densitometry, are observed. It is conceivable that these result from changes at only a subset of the phosphorylation sites minium Hours after shift Antisera to p8tdcl0 identify a protein in human cells Recently a human homologue of the cdc2+ gene of fission yeast (Lee & Nurse, 987; Draetta et al. 987) has been identified. We have used the antiserum to the p87 cdcl protein to see if it identifies any proteins in human cells. We performed a Western blot of proteins extracted from a 0 B' Fig. 3. p87 cdc0 protein level and phosphorylation during the cell cycle. Small cells were selected from an exponentially growing culture of weel-sq cells at 25 C using an elutriator rotor and shifted to 3 C. [32P]phosphate was added at this time. Samples were taken at intervals thereafter and protein was extracted for either Western blotting or immunoprecipitation with serum against p87 cdc0. A. Cell number after shift to 3 C. The bars indicate the times at which the protein samples were taken. Samples were taken at the same time as the cell number was determined. B. p87 cdcl protein level during the mitotic cell cycle. Protein samples were prepared at various times and transferred to nitrocellulose after electrophoresis. The Western blot was the probed sequentially with antiserum to p87 cdcl and 25 Ilabelled protein A. C. p87 ccicltl phosphorylation during the mitotic cell cycle. Protein samples were prepared for immunoprecipitation with antibody to p87c at various times from cells labelled with [ 32 P]phosphate. The dried gel was exposed for 0 days at 70 C with an intensifying screen. 32P-labelled samples were not taken until point 3 to allow the cells to become labelled for one full cycle V. Simanis and P. Nurse B Fig. 4. p87 cdcl protein level and phosphorylation state during exit from the cell cycle. 972h~ cells were grown to a density of 2 x l 0 m l ~ ', harvested, washed twice in nitrogenfree minimal medium and resuspended at the same cell density in medium without ammonium chloride. The cells were incubated at 32 C and samples withdrawn at time 0 and at 2, and 9h thereafter. A. A Western blot of protein samples taken at the indicated times probed with antiserum to lzs i-labelled protein A. p87 cduo f o l l o w e d b y B. Immunoprecipitates of p87cd prepared from cells labelled before and during the starvation with [3ZP]phosphate. The cell numbers at the indicated times were: t = 0, l - 8 x l 0 m r ' ; / = 2, 3 - l X l 0 m l " ' ; / =, 4-Sxl0 ml" ; t = 9, 4-5X0 ml".

5 Fig. 5. Western blot of human cells with antisera to p87 cdcl. Protein samples were prepared from 5. pombe 972h~ and five different human cell lines in exponential growth. The figure shows a Western blot of protein samples from these cells probed with antiserum to p87 cdc0. Cell lines are lane, RT4 cells; lane 2, J cells; lane 3, Alexander cells; lane 4, HT29 cells; lane 5, RT2 cells; lane, S. pombe 972h". The nature of the lower band identified in lanes 2 and 3 is unknown but could represent a breakdown product of the p92 protein. number of different human cell lines. In all cell lines tested, a protein of apparent molecular weight was identified (Fig. 5) and it is possible that it represents a human homologue of the cdclo+ protein. Discussion We have used antisera raised against a ccfc/0+-/}-galactosidase fusion protein to identify the protein product of the cdclo+ gene of the fission yeast S. pombe. The protein has an apparent molecular weight of , in good agreement with that predicted from the DNA sequence, (Aves et al. 985). The protein is phosphorylated on multiple serine residues and has an apparent isoelectric point of on two-dimensional gel electrophoresis. We have found that the steady-state level of the protein does not change significantly through the mitotic cycle, and that the protein remains phosphorylated throughout the cell cycle. These data demonstrate that execution of cdclo+ gene function during the cell cycle is not effected either by changes in the steady-state level of the protein or by grossly changing its state of phosphorylation. When cells were starved of nitrogen, causing them to exit from the mitotic cell cycle and accumulate in Gi before start, no significant change in protein level was seen. The p87cd protein remained phosphorylated although a slight decrease of approximately 2-5-fold was observed in the phosphorylation level. This is in contrast to the behaviour of p34 2, the protein encoded by the other known start gene cdc2+, which undergoes extensive dephosphorylation as cells cease to proliferate after nitrogen starvation (Simanis & Nurse, 98). As our phosphopeptide mapping has demonstrated that there are multiple sites of phosphorylation in p87 cdcl, it is possible that there are more subtle changes in phosphorylation during the cell cycle or as cells cease to proliferate. For example, changes at only a subset of the phosphorylation sites may modulate p87 cdcl function to effect the traverse of start. Increasing the level of cdcl0+ mrna in the cell leads to increased levels of p87 cdci in the cell and to a similar increase in the amount of phosphorylated p87 cdcl. This result demonstrates that the protein kinase(s) responsible for phosphorylating p87 cd are not limiting in activity. This is in contrast to the situation with p34 cdc2, where overproduction of the protein does not lead to a corresponding increase in the amount of phosphorylated p34 cdc2 in the cell (Simanis & Nurse, unpublished data). Raising the amount of p87 cdcl in the cell results in an increased cell size at division. It is possible that substantial increase in the level of p87 cdcl interferes with normal execution of cdcl0+ gene function, thereby delaying progress through the cell cycle. The identity of the kinase(s) that phosphorylate p87 cdcl is at present unclear. The p87 cdcl protein remains phosphorylated throughout the mitotic cell cycle. It is possible that this phosphorylation is related to p87 cdcl function rather than its temporal regulation through the cell cycle. For instance, it may enable p87 cdcl to reach the correct compartment in the cell or perhaps to oligomerize either with itself or with other proteins. The predicted cdclo+ protein has homology with the SWI gene of budding yeast. The SWJ gene is not required for cell cycle progress per se but is involved in the control of the periodic transcription of the HO gene during Gi and thereby the regulation of switching of mating type (Breeden & Nasmyth, 987), while cdcl0+ is involved in the traverse of start and entry into the mitotic cell cycle. Once the cdcl0+ function has been executed the processes leading to S phase are initiated, such as the periodic accumulation of histone transcripts prior to DNA synthesis. In cdclo* mutants blocked before start this does not occur, yet in mutants such as cdc22ts, which are blocked in Gi post-start, histone transcripts accumulate normally (Matsumoto et al. 987). Since SWI is known to regulate the transcription of Gi-specific genes and the cdcw+ gene function is also required for activating histone gene expression, it is tempting to speculate that the homology between the two proteins S. pombe cdclo pivtein 55

6 reflects a shared aspect of their function that is involved in activating specific gene transcription during the Gi phase of the cell cycle. In the case of SWI, HO gene transcription would be activated leading to mating type switching, while in the case of cdcj0 + the activated genes would be required for progress through the cell cycle into S phase. The cdc2 + gene function of fission yeast has also been found in human cells (Lee & Nurse, 987; Draetta et al. 987), raising the possibility that other cell cycle genes might also be conserved through evolution. We have identified a 92000M r protein present in all human lines tested using antisera to p87 cd. The similarity in size of this protein with p87 c suggests that there may also be a homologue to the cdclo + gene product in mammalian cells. We are grateful to Steve Aves, University of Exeter, UK, for plasmid TB2, to Jennifer Southgate, Tissue Interaction Laboratory, ICRF, for samples of human cell lines and to our colleagues in the Cell Cycle Control Laboratory for constructive criticisms of this manuscript. We are also indebted to the staff of the Animal Unit, ICRF, Clare Hall, who performed all the experimental work on animals and we thank Clare Middlemiss for typing this manuscript. This work was funded by the Imperial Cancer Research Fund. References AVES, S. J., DURKACZ, B. W., CARR, A. & NURSE, P. (985). Cloning, sequencing and transcriptional control of the Schizosaccharomvces pombe cdclo start gene. EMBOJ. 4, BEACH, D., DURKACZ, B. & NURSE, P. (982). Functionally homologous cell cycle control genes in budding and fission yeast. Nature, band. 300, BEACH, D. & NURSE, P. (98). High-frequency transformation of the fission yeast Schizosaccharomvces pombe. Nature, Land. 290, BEEMON, K. & HUNTER, T. (978). 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