The anaphase-promoting complex works together with the SCF complex for proteolysis of the S-phase cyclin Clb6 during the transition from G1 to S phase

Fungal Genetics and Biology 91 (2016) 6–19

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The anaphase-promoting complex works together with the SCF complex for proteolysis of the S-phase cyclin Clb6 during the transition from G1 to S phase Shiao-Yii Wu a,1, Vivian Jen-Wei Kuan b,1, Yao-Wei Tzeng d,1, Scott C. Schuyler b,c,⇑, Yue-Li Juang e,⇑ a

Master Program in Microbiology and Immunology, Tzu-Chi University, Hualien, Taiwan Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan Department of Colorectal Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan d Institute of Medical Sciences, Tzu-Chi University, Hualien, Taiwan e Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan b c

a r t i c l e

i n f o

Article history: Received 22 October 2015 Revised 11 March 2016 Accepted 15 March 2016 Available online 16 March 2016 Keywords: Anaphase-promoting complex/cyclosome Cdh1 Cyclin-dependent kinase G1-S phase transition

a b s t r a c t In Saccharomyces cerevisiae, the S-phase cyclin Clb6 is expressed shortly before the G1/S transition. It has been shown that in S phase the SCFCdc4 ubiquitin ligase controls Clb6 proteolysis, which requires cyclindependent kinases activity. A Clb6-3A mutant, bearing non-phosphorylatable mutations at S6A, T39A, and S147A, was observed to be hyperstabilized in S-phase but was unstable in mitosis. In this study, we found that the APCCdh1 form of the Anaphase-Promoting Complex (APC) was required for Clb6 proteolysis in both early and late G1. An in vitro ubiquitination assay confirmed that Clb6 is a substrate for APCCdh1. A KEN box and a destruction box in the Clb6N-terminus were identified. Mutations in the KEN box (mkb) and/or the destruction box (mdb) enhanced Clb6 stability in G1. Expression of Clb6mkd, bearing both mutations in the mkb and mdb, allowed cells to bypass the late G1 arrest caused by cdc4-1. This bypass phenotype was observed to depend upon CDK phosphorylation at residues S6, T39 and S147. Compared to Clb6, overexpression of Clb6ST, bearing all five mutations of S6A, T39A, S147A, mkb and mdb in combination, had a greater effect on promoting expression of Clb2 and S-phase entry, caused a greater G2 delay and a greater defect in cell division. Swe1 was also required for bud emergence when Clb6ST was overexpressed. Our observations suggest that both APCCdh1 and SCFCdc4-dependent proteolysis of Clb6 at the G1/S border are crucial for multiple cell cycle regulated events including proper expression of Clb2, the G1/S and G2/M cell cycle transitions and for proper completion of cell division at mitotic exit. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Cyclin-dependent kinase complexes are central regulators of cell cycle progression in eukaryotic cells. The budding yeast Saccharomyces cerevisiae, has three G1 cyclins (Cln1-3), two S-phase cyclins (Clb5-6) and four mitotic cyclins (Clb1-4) (Bloom and Cross, 2007). These cyclins associate with the cyclin-dependent kinase (CDK) Cdc28 to execute their specific functions in cell cycle ⇑ Corresponding author at: Chang Gung University, College of Medicine, Department of Biomedical Sciences, 259 Wen-Hwa 1st Road, Kwei-Shan, Taoyuan 333, Taiwan (S.C. Schuyler). Institute of Biomedical Sciences, Mackay Medical College, 46, Sec. 3, Jhong-Jheng Rd., Sanzhi District, New Taipei City 252, Taiwan (Y.-L. Juang). E-mail addresses: [email protected] (S.C. Schuyler), [email protected] (Y.-L. Juang). 1 Equal contribution. http://dx.doi.org/10.1016/j.fgb.2016.03.004 1087-1845/Ó 2016 Elsevier Inc. All rights reserved.

progression. Control of the timing of cyclin synthesis and proteolysis is important for a transition from one stage to the next stage of the cell cycle. Cyclin proteolysis is regulated by the ubiquitin–proteasome pathway. Cyclin is polyubiquitinated via ubiquitin E3 ligases and thereby targeted to the 26S proteasome for destruction. Both the SCF (Skp1/Cdc53/F-box protein) and the APC (AnaphasePromoting Complex or Cyclosome) are E3 ligases and regulators of cyclin proteolysis (Bloom and Cross, 2007). The SCF complex is required for proteolysis of Cln1-3 and Clb6, whereas the APC complex for promoting proteolysis of Clb1-5 (Bloom and Cross, 2007; Yaglom et al., 1995). Cdc20 and Cdh1 are substrate-specific activators of the APC (Schwab et al., 1997; Shirayama et al., 1999; Visintin et al., 1997). APCCdc20 regulates proteolysis of Clb5 whereas APCCdh1 controls proteolysis of the mitotic cyclins Clb1-4 (Bloom and Cross,

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2007). Cdh1 activity is regulated in a cell cycle-dependent manner (Visintin et al., 1998; Zachariae et al., 1998). Starting at the G1/S border, CDK activity is able to phosphorylate Cdh1 to prevent APCCdh1-dependent proteolysis of mitotic cyclins until a later stage of the cell cycle (Zachariae et al., 1998). In late mitosis, both the FEAR (Cdc14 early anaphase release) network and the mitotic exit network are initiated to free the phosphatase Cdc14 from the nucleolus, where the free Cdc14 removes phosphate groups from phosphorylated Cdh1 (Shou et al., 1999; Stegmeier et al., 2002; Visintin et al., 1999). In turn, APCCdh1 is activated to initiate proteolysis of mitotic cyclins in late mitosis and its activity persists until entry into S phase of the subsequent cell cycle (Huang et al., 2001; Visintin et al., 1998; Zachariae et al., 1998). In the budding yeast, DNA replication is mainly initiated by Clb5-6/Cdc28 (Epstein and Cross, 1992; Kühne and Linder, 1993; Schwob and Nasmyth, 1993). Although Clb5 and Clb6 appear nearly simultaneously in late G1, Clb6 rapidly disappears at the G1/S border whereas Clb5 persists until late mitosis (Jackson et al., 2006). Clb5 proteolysis is dependent upon two different mechanisms, an APC-dependent and an APC-independent mechanism, but is independent of SCF complex activity (Bloom and Cross, 2007; Sari et al., 2007). By contrast, Clb6 proteolysis requires the SCFCdc4 complex in a CDK-dependent manner (Jackson et al., 2006). The determinants for SCFCdc4-dependent proteolysis of Clb6 reside within its N-terminal 135 amino acid residues including three potential phosphorylation sites at S6, T39 and S147 (Jackson et al., 2006). Although a Clb6-3A protein, bearing mutations of S6A, T39A, and S147A, is hyperstabilized and persists beyond the G1/S transition, Clb6-3A protein is still degraded in mitosis when expressed under its own promoter (Jackson et al., 2006). This suggests that another mechanism other than the SCF pathway can also contribute to Clb6 proteolysis. Here, we present evidence that APCCdh1 is also required for Clb6 proteolysis during the transition from G1 to S phase and that APCCdh1-dependent Clb6 proteolysis at the G1/S border is crucial for timing of the G1/S transition and other later cell cycle events.

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2.3. Assay for half-life of Clb6-13myc fusion protein and its derivatives Cells were arrested in early G1 or in late G1 in medium containing 2% sucrose at the indicated temperatures, galactose was added to a final concentration of 3% for induction of Clb6-13myc for 40 min, and then glucose was added to a final concentration of 2% and cycloheximide to a final concentration of 1 mg/mL to shut off transcription and translation. Cell samples were collected at the indicated time points and analyzed by Western blotting and flow cytometric analyses. To obtain protein extracts, cells were broken by glass beads in a 2.5% SDS solution at 4 °C. Immunoblots were developed using ECL and exposed to X-ray film. Clb6-13myc was detected with 9E10 mouse monoclonal antibodies (Calbiochem Co.), Pds1-3HA with 12CA5 mouse monoclonal antibodies (Santa Cruz Co.), Clb2 with rabbit anti-Clb2 polyclonal antibodies (Santa Cruz Co.), and Cdc28 with goat anti-Cdc28 polyclonal antibodies (Santa Cruz Co.). For quantification in Figs. 2–6, the exposed X-ray film was scanned and the band density was determined by Quantity One 4.3.1 software (Bio-Rad Co). 2.4. Flow cytometry Analysis of DNA content by flow cytometry was performed as modified from the procedure described previously (Haase and Reed, 2002). Cells were fixed in 75% ethanol and kept at 4 °C for at least 12 h. After the fixed cells were treated with 1 mg/mL RNase A (Sigma Chemical Co.) in 50 mM Tris buffer (pH 7.4) at 37 °C overnight, cells were treated with 55 mM HCl containing 10 mg/mL pepsin (Sigma Chemical co.) at 37 °C for 30 min. Last, cells were washed with 50 mM Tris buffer (pH 7.4) three times, and then stained with 10 lM Sytox dye (Molecular Probes Co.) in 50 mM Tris buffer (pH 7.4) for 15 min. Stained cells were pulsesonicated for 5 s before analysis. 10,000 Sytox dye-stained cells per sample were analyzed on a FACScan cytometer using CellQuest software (Becton Dickinson Co.) or GalliosTM flow cytometer using Kaluza software (Beckman Coulter Co.). 2.5. Fluorescence microscopy

2. Materials and methods 2.1. Yeast strains, media and growth conditions The yeast strains used in this study are listed in the Supplementary Table S1. All strains except for SCSY51, SCSY730, SCSY732 and SCSY734 are derivatives of W303 (ade2-1 ura3-1 leu2-3, 112 his311, 15 trp1-1 can1-100). Yeast growth, preparation of media, and genetic manipulation were performed as described (Adams et al., 1997). To synchronize cells in late G1, cdc4-1 or cdc4-1 cdh1D cells were incubated at 37 °C, a non-permissive temperature for cdc4-1, until P90% of cells showed either multiple or elongated buds. To synchronize cells in early G1, cells were treated with 2 lM a-factor until >95% cells were unbudded with a shmoo-like shape. To release cells from early G1 arrest, cells were harvested by filtration, washed, and resuspended in the indicated fresh medium lacking a-factor.

2.2. Plasmid and strain constructions Techniques for making plasmid constructs of CLB6 or its derivatives were performed as described (Sambrook and Russell, 2001). All the primers used in making the plasmid constructs are listed in the Supplementary Table S2. All the constructs we obtained were confirmed by DNA sequencing. Details of the construction of each plasmid and each strain can be provided upon request.

To stain nuclei, cells were fixed with 3.7% formaldehyde in 1
PBS buffer for 30 min at room temperature, washed with 1
PBS buffer three times, and then stained with 2.5 lg/mL 40 ,60 diamidino-2-phenylindole (DAPI; Sigma Chemical Co.) in 1
PBS buffer. DAPI-stained cells were pulse-sonicated for 5 s before being observed under a Nikon E800 fluorescence microscope with a Nikon CoolPix995 digital camera (Nikon Instech Co.). 2.6. Making large-scale yeast extracts Large-scale yeast cultures were grown and harvested as previously described (Schuyler and Murray, 2009). The yeast pellets from 80 °C were slightly defrosted on ice and the bottom of the 50 mL Falcon tubes were sliced off. The partially frozen pellets were pushed out into a 20 mL Bead-Beater chamber (Biospec Inc. OK). Glass beads were added to fill up about half the chamber. The chamber was filled with Lysis buffer (200 mM HEPES at pH 8.0, 150 mM NaCl, 10% (v/v) glycerol, 5 mM EDTA, 5 mM EGTA, 0.1% (v/v) NP-40, 1 mM NaVO4, 10 mM NaF, 5 mM sodium pyrophosphate, 10 mM b-glycerol phosphate, 1 mM DTT). 0.00348 g of PMSF was dissolved in 200 lL of 100% ethanol and added fresh to the lysis buffer directly before use. 1 mL of 1 mg/mL Pepstatin A was dissolved in methanol and added fresh to the lysis buffer directly before use. 1 protease inhibitor cocktail tablet (Complete, Roche, IN) was added. 5–7 bead-beating cycles of 30 s ‘‘on” and 2 min ‘‘off” were performed. The lysate was poured into a 50 mL Falcon tube and centrifuged at 10,000g for 10 min

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Fig. 1. Overexpression of Clb6 in both CDC14 and cdc14-1 cells at the permissive temperature. Both CDC14 and cdc14-1 cells bearing either the GAL1 vector or GAL1-CLB613myc construct were incubated in YEP + 2% sucrose medium overnight, and then diluted to an OD600nm of 0.2 in YEP + 2% galactose for induction of Clb6-13myc at 25 °C. Cells were collected at the indicated times for fluorescence microscopic and Western blot analyses. Three independent experiments were performed. (A) Chained cells. At 8 h after induction of Clb6-13myc in YEP + galactose medium, cells were collected, fixed with formaldehyde, and then stained with DAPI for observation of nuclei. Chained cells with three, four, or more cellular units are shown. Size of scale bar is 5 lm. (B) Levels of Clb6 in CDC14 and cdc14-1 cells. Western blots were performed to detect levels of Clb6-13myc and Cdc28. 9E10 mouse monoclonal antibodies were used to probe Clb6-13myc and goat anti-Cdc28 polyclonal antibodies Cdc28. Levels of Cdc28 were used as a loading control.

Fig. 2. Clb6 has an enhanced stability in cdc16-123 early G1 cells at the non-permissive temperature. CDC16 and cdc16-123 strains bearing the GAL1-CLB6-13myc construct were arrested with a-factor in YEP + sucrose medium at 25 °C, shifted to 37 °C for 30 min, and then galactose was added to a final concentration of 3% for induction of Clb6 for 40 min. After induction of Clb6, 2% glucose and 1 mg/mL cycloheximide were added to turn off CLB6 transcription and translation. The zero time point is when glucose and cycloheximide were added. Cells were collected at the indicated times for flow cytometric and Western blot analyses. (A) Levels of Clb6 and Cdc28. Proteins were detected as described in Fig. 1B. (B) Quantification of protein levels. Band intensity shown in (A) was determined by Quantity One 4.3.1 software (Bio-Rad, Co). Clb6 intensity was normalized to Cdc28 intensity. The values at each of the indicated time points in the graph are the means ± s.d. from two independent experiments. Clb6-13myc had a higher stability in cdc16-123 cells than in CDC16 cells with a p value of <0.001. The p values were obtained by the Two-way ANOVA with repeated measures. (C) Flow cytometric analysis for confirmation of early G1 arrest.

at 4 °C. The supernatant was poured into a new 50 mL Falcon tube. This crude extract was aliquoted into microcentrifuge tubes of 1 mL each and store at 80 °C for further use. 2.7. Preparation of

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S-methionine labeled Clb6 substrate

Using a T7 coupled reticulocyte lysate system using S-methionine, Clb6 proteins were isotopically-labeled. This allows for phosphor-imaging after running SDS–PAGE (Schuyler and Murray, 2009). To a final volume of 500 lL, 400 lL of TnT T7 Quick Master Mix (Promega, USA), 20 lL of 35S-methionine and 10 lg of plasmid DNA were added following the manufactures recommendations. The extra volume was filled up using nuclease-free water. The reaction was allowed to proceed for 2 h at room temperature.

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2.8. Kinase assays TAP-tagged Pho85, Cdc28, Clb5, Clb6, Cln1, Cln2, Pcl1 or Pho80 extracts from cdc4-1 arrested cells were defrosted. Pan Mouse IgG Dynabeads (Invitrogen, USA) were washed with 1 mL of ddH2O and 2 mL of wash buffer. Wash buffer contained 200 mM HEPES at pH 8.0, 150 mM NaCl, 10% glycerol. TAP-tagged Pho85, Cdc28, Clb5 Clb6, Cln1, Cln2, Pcl1 or Pho80 were allowed to bind to IgG beads for 2 h at 4 °C. Beads are washed with 1 mL wash buffer and 2 mL kinase assay buffer (20 mM HEPES at pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 20 mM beta-glycerol phosphate, 0.1 mM NaVO4, 1 mM NaF and 0.05 mM ATP) before being added to kinase assays. 2 lL of 35S-methionine labeled purified Clb6 was added. Reactions were incubated for 1 h at room temperature.

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Fig. 3. Clb6 has an enhanced stability in cdc4-1 and cdh1D early G1 cells at the nonpermissive temperature. (A–C) Wild type, cdc4-1, cdh1D, and cdc4-1 cdh1D strains bearing the GAL1-CLB6-13myc construct were used to determine Clb6 stability. The experiments and analyses were performed as described in Fig. 2. Two independent experiments were performed. (A) Levels of Clb6 and Cdc28. (B) Flow cytometric analysis for confirmation of early G1 arrest. (C) Quantification of protein levels. Band intensity shown in (A) was determined by Quantity One 4.3.1 software (Bio-Rad, Co). Clb6 intensity was normalized to Cdc28 intensity. (D) APCCdh1 assays 35S-Methionine labeled wild-type Clb6. APC was prepared from cdc4-1 arrested yeast lysates. Co-factor Cdh1 was prepared fresh by IVT/T. (E) Quantification of unmodified Clb6 substrate remaining over time, where p-value < 0.05 by Student’s t-test at 60 min.

2.9. In vitro transcription/translation of Cdh1

3. Results

Using a T7 coupled reticulocyte lysate system and providing methionine, the co-factor Cdh1 was made from the prepared plasmid DNA, which has a T7 promoter site (Schuyler and Murray, 2009). For a final volume of 20 lL, 16 lL of TnT T7 Quick Master Mix, 0.4 lL of methionine and 0.4 lg of plasmid DNA were added. The extra volume was filled up using nuclease-free water. The reactions were allowed to proceed for 2 h at room temperature.

3.1. The APC is required for Clb6 proteolysis in early G1

2.10. APCCdh1 Assays TAP-tagged Cdc16 arrested cell extracts were defrosted. Pan Mouse IgG Dynabeads were washed with 1 mL of ddH2O and 2 mL of wash buffer. Wash buffer contained 200 mM HEPES at pH 8.0, 150 mM NaCl, 10% glycerol. APC was allowed to bind to IgG beads for 2 h at 4 °C. Beads were washed three times with 1 mL wash buffer each before being added to enzyme assay. The assay mixture contained ubiquitin, an ubiquitin-activating enzyme E1, an ubiquitin-conjugating enzyme E2, an ubiquitin ligase APC, Cdh1, ATP, ubiquitin aldehyde, QAH buffer and the appropriate Clb6 substrate (Schuyler and Murray, 2009).

A previous study had shown that cdc14-1 cells arrest in telophase with high levels of Clb2 at the nonpermissive temperature, and overexpression of Clb2 prevents growth of cdc14-1 cells at the permissive temperature (Jaspersen et al., 1998). Therefore, we sought to examine whether overexpression of Clb6 had the same effect on the growth of cdc14 mutants as that of Clb2. To test this, CLB6 was fused at its C terminus to a 13-myc tag and expressed by the GAL1 promoter, where the resultant construct (GAL1-CLB6-13myc) was integrated into the LEU2 locus of both CDC14 and cdc14-1 strains. Overexpression of Clb6 caused a growth-retardation in both CDC14 and cdc14-1 cells after 2-days of incubation on galactose medium plates at 25 °C, a permissive temperature for cdc14-1 cells (our observations). However, overexpression of Clb6 resulted in an accumulation of cellular chains (12%) in cdc14-1 cells after an 8-h incubation at 25 °C (Fig. 1A and Supplementary Table S3). Overexpression of untagged Clb6 also resulted in an accumulation of chained cells in cdc14-1 cells (Supplementary Table S4). This indicated that the phenotype of chained cells did not result artificially from the myc tag. We then examined whether the formation of chained cells was accompa-

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Fig. 4. Both a KEN box and a D-box are required for Clb6 proteolysis in early G1. Wild type strains bearing GAL-CLB6-13myc, GAL1-clb6mkb-13myc, GAL1-clb6mdb-13myc, or GAL1-clb6mkd-13myc constructs were used for analyses. The experiments were performed as described in Fig. 2 except that yeast strains were incubated at 30 °C. (A) Sequence of N-terminal 150 amino acids of Clb6. Both the KEN box and the destruction box are underlined and the conserved amino acid residues are in bold face. The amino acid residues S6, T39, and S147 are also in bold face. (B) Levels of Clb6 and Cdc28 proteins. (C) Quantification of protein levels. Band intensities shown in (B) were determined by Quantity One 4.3.1 software (Bio-Rad, Co). The values at each of the indicated time points in the graph are the means ± s.d. from three independent experiments. Clb6mkb13myc, Clb6mdb-13myc, and Clb6mkd-13myc had a higher stability than Clb6-13myc with respective p values of <1
105. The p values were obtained by the Two-way ANOVA with repeated measures. (D) Flow cytometric analyses to confirm an early G1 arrest.

nied by an accumulation of higher levels of Clb6 protein. We found that much higher protein levels of Clb6 were observed in cdc14-1 cells than in CDC14 cells after 4 h of incubation (Fig. 1B). Therefore, high accumulation of Clb6 protein and formation of chained cells was due to an effect of the cdc14-1 mutation. Cdc14 dephosphorylates Cdh1 to allow Cdh1to bind and activate APC in late mitosis (Visintin et al., 1998). Thus, our observation prompted us to consider that Clb6 proteolysis could also be APC-dependent, and hence much higher protein levels of Clb6 could accumulate in cdc14-1 cells but not in CDC14 cells. Jackson et al. has shown that the SCFCdc4 complex, not the APC, promoted Clb6 proteolysis in S phase (Jackson et al., 2006), but they did not investigate the protein stability in mitosis or in G1 phase. To test our hypothesis, we examined Clb6 stability in early G1 when APCCdh1 is known to be active. Cdc16 is an essential component of APC and its defect will enhance the stability of an APC substrate (Irniger et al., 1995). Thus, the cdc16-123 temperature-sensitive mutant strain was used for our analyses (Irniger et al., 1995). Cells were arrested in early G1 by the addition of a-factor. If Clb6 was a substrate of APC, we expected to observe that Clb6 would become more stable in cdc16-123 early G1 cells. By performing the GAL1 promoter shutoff experiment as described in the legend to Fig. 2, the half-life of Clb6 was measured in both CDC16 and cdc16-123 early G1 cells at 37 °C, a non-permissive temperature for cdc16-123 mutation. The half-life of Clb6 was 10–12 min in cdc16-123 early G1 cells whereas much less than 3 min in CDC16 early G1 cells, indicating that stability of Clb6 was enhanced by loss of Cdc16 functions (Fig. 2). This observation suggests that APC plays a role in controlling Clb6 stability in G1.

cdc20-1 and cdh1D mutant strains were used. We first examined Clb6 stability in CDC20 and cdc20-1 early G1 cells at 37 °C, a nonpermissive temperature for the cdc20-1 mutation. We observed that Clb6 stability was not significantly different (Supplementary Fig. S1). This indicated that Clb6 proteolysis did not depend upon Cdc20. Clb6 stability was then examined in CDH1 and cdh1D cells in early G1 at 37 °C. We observed that in cdh1D early G1 cells Clb6 stability was enhanced, where the half-life of Clb6 was 6–8 min (Fig. 3A–C). In CDH1 early G1 cells the half-life of Clb6 was much less than 2 min, indicating that Clb6 proteolysis required Cdh1 (Fig. 3A–C). We also determined whether the SCFCdc4 complex was also required for Clb6 proteolysis in early G1, where cdc4-1 cells were used for the analyses. Consistent with previous observations, Clb6 was more stable and had a half-life of 3–4 min in cdc41 early G1 cells at 37 °C (Fig. 3A–C). Since both Cdh1 and Cdc4 contributed to Clb6 proteolysis in early G1 we predicted that cdc4-1 cdh1D double mutations should have a more pronounced effect on Clb6 stability in early G1 at 37 °C. As expected, Clb6 had a half-life of 36–38 min, and Clb6 was more stable in cdc4-1 cdh1D early G1 cells than in both cdc4-1 and cdh1D early G1 cells at 37 °C (Fig. 3A–C). This indicates that Clb6 proteolysis requires both the SCFCdc4 and the APCCdh1 in early G1. To confirm the in vivo observation that Clb6 stability is regulated by the APC, an in vitro APCCdh1 assay was performed (Fig. 3D and E). A weak level of consumption of the full-length Clb6 protein substrate by the APCCdh1 enzyme activity was observed.

3.2. Cdh1, but not Cdc20, is required for Clb6 proteolysis in early G1

KEN box and the D-box (destruction box) sequences are the motifs recognized by the APC (Peters, 2006). The conserved sequence of the KEN box is K–E–N and that of a D-box is R–x–x– L–x–x–x–x–N. Previous studies showed that the most divergent

To determine whether APC-dependent proteolysis of Clb6 required either the substrate-specific activator Cdc20 or Cdh1,

3.3. Both N-terminal KEN and destruction boxes are determinant motifs required for Clb6 proteolysis

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Fig. 5. Cdh1 is required for Clb6 proteolysis in late G1. cdc4-1 strains bearing GAL-CLB6-13myc or GAL1-clb6mkd-13myc constructs and cdc4-1 cdh1D strain bearing the GALCLB6-13myc construct were used to examine Clb6 stability in late G1. The experiments and analysis were performed as described in Fig. 2 except that cells were arrested in late G1 without the addition of a-factor in YEP + sucrose medium at 37 °C. (A) Levels of Clb6 and Cdc28 proteins. (B) Quantification of protein levels. Band intensities shown in (A) were determined by Quantity One 4.3.1 software (Bio-Rad, Co). The values at each of the indicated time points in the graph are the means ± s.d. from three independent experiments. Clb6-13myc in cdc4-1 cdh1D cells and Clb6mkd-13myc in cdc4-1 cells had a higher stability compared to Clb6-13myc in cdc4-1 cells with respective p-values of <0.001 and <0.01. The p-values were obtained by Two-way ANOVA with repeated measures. (C) Flow cytometric analyses for confirmation of the late G1 arrest.

Fig. 6. Clb6 stability in late G1 cells when expressed by the CLB6 promoter. cdc4-1 cells bearing the CLB6-13myc construct and cdc4-1 cdh1D cells bearing CLB6-13myc construct were diluted to an OD600nm of 0.07 after overnight incubation in YEP + glucose medium at 25 °C. Cells were then incubated at 37 °C until P90% of cells showed either multiple or elongated buds. Cells were subsequently treated with 1 mg/mL cycloheximide. The zero time point indicates when cycloheximide was added. Cells were collected at the indicated times for flow cytometric and Western blot analyses. (A) Levels of Clb6 and Cdc28 proteins. Western blots were performed to detect levels of Clb613myc and Cdc28 as described in Fig. 1. (B) Quantification of protein levels. Band intensities shown in (A) were determined by Quantity One 4.3.1 software (Bio-Rad, Co). The values at each of the indicated time points in the graph are the means ± s.d. from four independent experiments. Clb6-13myc had a higher stability in cdc4-1 cdh1D cells than in cdc4-1 cells with a p value of <0.001. The p values were obtained by the Two-way ANOVA with repeated measures. (C) Flow cytometric analyses for DNA content of cells.

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region of Clb5 and Clb6 protein sequences resides within the Nterminus and that deletion of the N-terminal 100 or 135 amino acid residues enhanced Clb6 stability (Jackson et al., 2006). Clb5 has a D-box targeted by the APC in its N-terminus (Irniger et al., 1995; Wasch and Cross, 2002). We reasoned that either a KEN box or D-box might also reside within the N-terminal 135 amino acids of Clb6. We found that Clb6 had a putative KEN box from the amino acid residues 23–25 and a putative D-box sequence (RGKLQRDST) from the amino acid residues 64–72 (Fig. 4A). To determine whether the putative KEN box was contributed to Clb6 stability, lysine 23, glutamic acid 24, and asparagine 25 in the KEN box were changed to alanine to make CLB6mkb mutant. We then measured the half-life of Clb6mkb in early G1 cells at 30 °C. We found that Clb6mkb was more stable than Clb6 in early G1 with a half-life of 7–9 min whereas Clb6 had a half-life of 2–4 min (Fig. 4B–D). Likewise, to determine whether the putative D-box contributed to Clb6 proteolysis, arginine 64 and leucine 67 in the putative D-box were changed to alanine to make CLB6mdb mutant. The Clb6mdb had a half-life of 10–12 min and was also more stable in early G1 than Clb6 (Fig. 4B–D). We further investigated whether the Clb6mkd protein bearing mutations in KEN box and D-box was more stable than both Clb6mkb and Clb6mdb in early G1. Unexpectedly, Clb6mkd was as stable as Clb6mdb, but a little bit more stable than Clb6mkd, indicating that simultaneous mutations in both KEN box and D-box did not have an additive effect on Clb6 stability at least under our conditions (Fig. 4B–D). It is possible that SCFCdc4-dependent pathway may counteract the effect of mutations in both KEN box and Dbox on Clb6 proteolysis in early G1. Despite this, both the KEN box and the D-box sequences appear to be the determinants making a contribution to Clb6 proteolysis. 3.4. APCCdh1 is required for Clb6 proteolysis in late G1 It is known that APCCdh1 is gradually inactivated and its persistent activity is able to promote proteolysis of Cdc20 and Ase1, known substrates of APCCdh1, but not of Clb2 in late G1 (Huang et al., 2001). Thus, we asked whether the persistent APCCdh1 activity contributed to Clb6 proteolysis in late G1. Because the cdc4-1 mutation can result in a cell cycle arrest in late G1 at 37 °C, we determined Clb6 stability in cdc4-1 and cdc4-1 cdh1D cells in late G1. The half-life of Clb6 was 22–25 min in cdc4-1 cdh1D late G1 cells and 7–10 min in cdc4-1 late G1 cells (Fig. 5). Therefore, like Ase1 and Cdc20 (Huang et al., 2001), Clb6 was more stable in cdc4-1 cdh1D late G1 cells than in cdc4-1 late G1 cells. We also examined Clb6mkd stability in cdc4-1 late G1 cells. Consistently, Clb6mkd had a half-life of 12–15 min and was more stable than Clb6 in cdc4-1 late G1 (Fig. 5). Taken together, we conclude that the persistence of APCCdh1 activity and both the KEN box and Dbox are required for proteolysis of Clb6 in late G1. Notably, our results revealed that Clb6 was more stable in cdc41 cdh1D cells than in cdc4-1 cells, but still not quite as stable in cdc4-1 cdh1D cells in late G1 (Fig. 5). Because cdc4-1 and cdh1D mutations could inactivate the SCFCdc4 and the APCCdh1 complexes respectively, this instability indicated that at the G1/S transition Clb6 proteolysis was also regulated by some mechanism(s) other than the SCFCdc4 and the APCCdh1 ubiquitination pathways. It is also possible that some mechanism(s) other than both the SCFCdc4 and the APCCdh1 ubiquitination pathways are not relevant when Clb6 protein is expressed at a normal level. To clarify this, we also examined stability of Clb6 and Clb6mkd in CDC4, cdc4-1 or cdc4-1 cdh1D cells when expressed from the CLB6 promoter (Supplementary Fig. S2). Cells were incubated for 40 min at 37 °C and then treated with cycloheximide to determine Clb6 stability. Similarly, Clb6 was highly unstable in CDC4 cells but its stability was enhanced in cdc4-1 cells and was also more stable

in cdc4-1 cdh1D cells than in cdc4-1 cells (Supplementary Fig. S2). Clb6mkd was more stable than Clb6 in cdc4-1 cells. Nevertheless, Clb6 was still not quite stable in cdc4-1 cdh1D cells (Supplementary Fig. S2). To further confirm that APCCdh1 promotes degradation of the natively expressed Clb6 proteins at the G1/S border, we investigated Clb6 stability in cdc4-1 and cdc4-1 cdh1D cells arrested in late G1 when expressed from the CLB6 promoter. We found that the half-life of Clb6 was 18–22 min in cdc4-1 cdh1D cells and less than 10 min in cdc4-1 cells (Fig. 6). Therefore, consistent with the above observations in late G1 (Fig. 5), Clb6 proteins under the native promoter were also more stable in cdc4-1 cdh1D cells than in cdc4-1 cells. These observations support the hypothesis that APCCdh1 is required for degradation of Clb6 proteins at the G1/S border. Additionally, the Clb6 proteins were still degradable in cdc4-1 cdh1D cells (Fig. 6), indicating that some mechanism(s) other than the SCFCdc4 and the APCCdh1 pathways may also regulate turnover of Clb6 in late G1. 3.5. Clb6 is a direct substrate of late G1 cyclin-dependent kinases Cdc28 and Pho85 Previous work had shown Clb6 is either a direct or indirect substrate of Cdc28 and Pho85 in vivo in S phase (Jackson et al., 2006). We investigated if Clb6 can be a direct substrate of Cdc28 and/or Pho85 isolated from G1-arrested cells in vitro. 35S-methionine labeled wild-type Clb6 and mutant Clb6-3A proteins were prepared by in vitro transcription/translation (IVT/T). These pure proteins were used as substrates in kinase assays. Kinase assays were performed in triplicate for wild-type Clb6 and mutant Clb6-3A proteins (Fig. 7A and B). Both Cdc28 and Pho85 isolated from cdc4-1 arrested cells were able to phosphorylate wild-type Clb6, as indicated by gel shifts. Neither Cdc28 nor Pho85 were able to phosphorylate clb6-3A, where the three possible phosphorylation sites S6, T39 and S147 had been mutated to alanine. In addition, Clb5 and Clb6 isolated from cdc4-1 arrested cells, where it is predicted their activity is still inhibited by Sic1, were not able to phosphorylate Clb6 (Fig. 7A and B). To determine if Cdc28-dependent phosphorylation depends upon Cln activity, Cln1 and Cln2 were isolated from cdc4-1 arrested cells and it was observed that they are also functional for phosphorylating Clb6 (Fig. 7E and F). These observations demonstrate that Clb6 is a direct substrate for Cdc28 and Pho85 in vitro. To narrow down which of the three phosphorylation sites are targets of Cdc28 and Pho85 in vitro, mutant alleles of Clb6 with single, double, and triple phosphorylation site mutations were employed as target substrates in the kinase assays. Our results showed that single mutant alleles in Ser6 or Thr39 were still phosphorylated by Cdc28 and Pho85, although not as strongly as wildtype, whereas mutation in Ser147 prevented the phosphorylation (Fig. 7C and D). Kinase assays using Cln1 and Cln2 gave similar results as Cdc28 (Fig. 7E and F). Pcl1 and Pho80, cyclin partners for Pho85, isolated from cdc4-1 cells were not able to promote phosphorylation of Clb6 in vitro (Supplementary Fig. S3). In combination, Clb6 is a direct substrate of the late G1 cyclin-dependent kinases Cdc28 and Pho85 where Ser147 is the most prominent target site. 3.6. Clb6am, a mutant protein bearing mutations of mkb, mdb and S147A partially relieves the late G1 arrest caused by cdc4-1 It is puzzling as to why Clb6 expression persists in late G1. Since Clb6 proteolysis is both APC and SCFCdc4-dependent in late G1, we speculated that both APC and SCFCdc4-dependent Clb6 proteolysis is required to control the timing of G1-S transition. To test this, we made a stabilized version of CLB6 mutant (clb6ST) construct

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Fig. 7. Clb6 is a direct substrate of the late G1 cyclin-dependent kinases Cdc28 and Pho85. (A and B) Kinase assays on 35S-Methionine labeled wild-type and mutant Clb6. Phosphor-images of TAP-tagged Pho85, Cdc28, Clb5 and Clb6 isolated from cdc4-1 arrested cells used to phosphorylate 35S-Methionine labeled wild-type and mutant clb6-3A proteins. Three independent kinase assays were performed for Pho85, Cdc28 and Clb5, whereas two independent assays were performed for Clb6. (C and D) Kinase assays on 35 S-Methionine labeled wild-type, single mutation, double mutations and triple mutations Clb6 with Cdc28 and Pho85 from cdc4-1 arrested cells. (E and F) Kinase assays on 35 S-Methionine labeled wild-type, single mutation, double mutations and triple mutations Clb6 with Cln1 and Cln2 from cdc4-1 arrested cells.

bearing mutations of S6A, T39A, S147A, mkb and mdb for analysis. Since our observations also revealed that CDK mainly phosphorylated the residue at S147, and only slightly at residues S6 and T39 (Fig. 7), we also made the clb6 mutant bearing mutations atS147A, mkb and mdb designated as clb6am. We then determined whether untagged Clb6mkd, Clb6S147A, Clb6-3A, Clb6ST or Clb6am could make cells bypass the late G1 arrest caused by cdc4-1 when expressed under the native CLB6 promoter. After incubation at 37 °C for 6 h, flow cytometric analyses were performed to determine the DNA content of cdc4-1 cells bearing CLB6 or the mutant alleles. We found that 86% of cdc4-1 CLB6 cells, cdc4-1 clb6mkd cells, cdc4-1 clb6S147A cells, cdc4-1 clb6-3A cells, and cdc4-1 clb6ST cells had a DNA content of 1C (Fig. 8A), indicating that like Clb6, Clb6S147A, Clb6-3A, Clb6mkd, and Clb6ST could not relieve the late G1 arrest caused by cdc4-1. However, 62% of cdc4-1 clb6am cells had a DNA content of 1C and 32% of cdc4-1 clb6am cells a DNA content of 2C (Fig. 8A). This indicated that Clb6am could partially relieve the late G1 arrest caused by cdc4-1. It is known that the cdc4-1 mutant cells arrest in late G1 and form multiple buds (designated bud projections here) at the nonpermissive temperature (Schwob et al., 1994; Singer et al., 1984). We also determined whether Clb6mkd, Clb6S147A, Clb6-3A, Clb6am or Clb6ST could suppress the formation of projections in cdc4-1 cells at the non-permissive temperature. After cells were incubated at 37 °C for 4 h, we found that 77% of cdc4-1 CLB6 cells and 76%

of cdc4-1 clb6S147A cells were budded with projection(s), whereas 64% of cdc4-1 clb6mkd, 52% of cdc4-1 clb6-3A, 65% of cdc4-1 clb6am, and 48% of cdc4-1 clb6ST cells displayed projections (Fig. 8B). These observations indicate that cdc4-1 clb6S147A cells behaved similarly as cdc4-1 CLB6 cells, and that Clb6-3A and Clb6ST can exert an effect on suppressing formation of cells with bud projection(s) suggesting that CDK-dependent phosphorylation may regulate function of Clb6. 3.7. A stabilized version of Clb6 induces formation of chained cells Since our observations have shown that overexpression of Clb6 could induce formation of cellular chains in cdc14-1 cells, we asked whether overexpression of Clb6-13myc, Clb6mkd-13myc, Clb6-3A13myc or Clb6ST-13myc could cause an accumulation of chained cells in an otherwise wild type cell cultures. At 8 h after galactose was added for induction of Clb6 or its derivatives at 30 °C, 12% of cells overexpressing Clb6ST-13myc were in a chained form, whereas less than 4% of cells overexpressing Clb6-13myc, Clb6mkd13myc or Clb6-3A-13myc, and none of cells bearing the GAL1 vector were in a chained form (Table 1). This indicates that overexpression of Clb6ST resulted in a significant increase in number of chained cells. Of note, the protein level of Clb6ST-13myc was similar to that of Clb6-3A-13myc but higher than that of Clb6-13myc and Clb6mkd-13myc (Supplementary Fig. S4). Taken together, we

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Fig. 8. Effect of Clb6 and its derivatives on the late G1 arrest induced by cdc4-1. (A) Determination of DNA contents of cdc4-1 CLB6 cells and its derivatives. cdc4-1 CLB6, cdc4-1 clb6S147A, cdc4-1 clb6-3A, cdc4-1 clb6mkd, cdc4-1 clb6am, and cdc4-1 clb6ST cells were incubated for 6 h at 25 °C or 37 °C. Cells were then fixed with ethanol, treated with RNase, and stained with sytox dye. DNA contents were measured by flow cytometry. (B) Morphology of cdc4-1 CLB6 cells and its derivatives at the nonpermissive temperature. cdc41 CLB6, cdc4-1 clb6S147A, cdc4-1 clb6-3A, cdc4-1 clb6mkd, cdc4-1 clb6am, and cdc4-1 clb6ST cells were incubated for 4 h at 37 °C before being fixed with 3.7% formaldehyde. Two hundred cells were scored for each of three independent experiments. Values in the graph are the mean ± s.d. from three independent experiments. The p-values for formation of budded projections in different strains of cell culture (compared to cdc4-1 CLB6 cells) were obtained by the Student’s t-test. ⁄, p < 0.001; ⁄⁄, p = 0.125.

Table 1 Effect of CLB6 mutations on formation of chained cells. Strains

Chained cells (%)

pGAL1 pGAL1-CLB6-13myc pGAL1-clb6mkd-13myc pGAL1-clb6-3A-13myc pGAL1-clb6ST-13myc

0 1.59 ± 0.23 (p < 0.0005) 2.89 ± 0.53 (p < 0.001) 3.12 ± 1.04 (p < 0.001) 11.74 ± 0.45 (p < 0.005)

Wild type cells bearing the indicated construct were incubated in the galactose medium starting with an OD600nm of 0.3 for 8 h at 30 °C. Values are means ± s.d. from three independent experiments. 200 cells were counted for each of three independent experiments.

conclude that overexpression of Clb6ST significantly induced formation of chained cells in wild type cell culture. 3.8. A stabilized version of Clb6 promotes premature accumulation of Pds1 and Clb2, causes a premature G1/S transition and induces a significant delay in G2 Based upon previous observations (Jackson et al., 2006) and our current observations, it is known that Clb6 proteolysis is at least regulated by both the APCCdh1 and the SCFCdc4 complexes at the G1/S border. Previous studies revealed that overexpression of Clb6 can induce a G2 delay in cell cycle progression (Basco et al.,

1995). We asked what other stages of cell cycle were affected by overexpression of Clb6ST. Cells bearing GAL1 PDS1-3HA, GAL1CLB6-13myc PDS1-3HA or GAL1-clb6ST-13myc PDS1-3HA were arrested in early G1 in sucrose medium by the addition of afactor. After arrest, Clb6-13myc or Clb6ST-13myc was expressed by the addition of galactose in early G1 cells for 30 min and cells were then released into the galactose medium lacking a-factor. At 150 min after release from G1, a-factor was added to make cells arrest in G1 of the subsequent cell cycle. Samples were taken every 30 min for microscopic, flow cytometric and Western blot analyses. At 60 min after release from G1 arrest, 40% of cells expressing Clb6-13myc and 40% of cells expressing Clb6ST-13myc were budded whereas only 20% of cells bearing the GAL1 vector were budded (Fig. 9A–C). Flow cytometric analysis also revealed that at 60 min after release from G1 arrest, 15% of cells expressing Clb6ST-13myc and 28% of cells expressing wild type Clb613myc had a DNA content of 1C whereas 43% of cells bearing the GAL1 vector still had a DNA content of 1C (Fig. 9D–F). Therefore, overexpression of Clb6-13myc and Clb6ST-13myc accelerated bud emergence, but overexpression of Clb6ST-13myc promoted S-phase entry more significantly than that of Clb6-13myc. Previous observations had shown that overexpression of wild type Clb6 could cause a delay in G2 and thereby inhibiting or delaying nuclear separation. This is also the case in our observations. At 240 min after release from G1 arrest, only 1% of budded

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Fig. 9. Effect of CLB6 mutation on cell cycle progression. Strains bearing the PDS1-3HA and the GAL1 (A, D, G), GAL1-CLB6-13myc (B, E, H), or GAL1-clb6ST-13myc (C, F, I) constructs were used for analyses. Cells were diluted to an OD600nm of 0.1 in 250 mL of YEP + sucrose medium, and then arrested with a-factor at 30 °C. CLB6 expression was induced with the addition of galactose for 30 min. Cells were collected by filtration, washed and released into 250 mL of fresh YEP + galactose medium lacking a-factor (aF). The arrows indicate that at 150 min after release from G1 arrest, a-factor was added to make cells arrest in G1 of the subsequent cell cycle. Cells were collected at indicated times for DAPI-staining, flow cytometric and Western blot analyses. (A–C) Observing the morphology and nucleus of cells. Cells were fixed with ethanol, and then stained with DAPI for observation of the nuclei. Two hundred cells were counted for each time point. Open circle (s) unbudded cells with one nucleus; closed circle (d) budded cells with one nucleus; open square (h) budded cells with two nuclei; closed square (j) chained cells. (D–F) Determination of DNA content of cells. Cells were fixed with ethanol, treated with RNase and stained with sytox dye. DNA content was measured by flow cytometry. (G–I) Determination of the protein levels of Clb6, Pds1, Clb2 and Cdc28. Detection of Clb6 and Cdc28 was performed as described in Fig. 1. Pds1-HA was detected with 12CA5 mouse anti-HA monoclonal antibodies and Clb2 with rabbit anti-Clb2 polyclonal antibodies. No gal indicates cell lysates from cells that were arrested in early G1 and collected before the addition of galactose.

cells bearing the GAL1 vector left had an undivided nucleus whereas 25% of budded cells overexpressing Clb6-13myc and 50% of budded cells overexpressing Clb6ST-13myc still bore an undivided nucleus (Fig. 9A–C). This indicated that Clb6ST-13myc caused a greater delay in G2 or nuclear separation than wild type Clb6-13myc when overexpressed. Pds1 (securin in mammals) binds and inhibits separase to prevent cleavage of cohesins, which hold together the paired sisterchromatids before anaphase (Ciosk et al., 1998; Cohen-Fix et al., 1996). Pds1 proteolysis frees separase, which in turn cleaves cohesins to initiate separation of the paired sister-chromatids and nuclear separation (Ciosk et al., 1998). Thus, we also examined the

protein levels of Pds1-3HA and Clb2. At 30 min after release from G1 arrest, the protein level of Pds1-3HA was higher in cells overexpressing Clb6 or Clb6ST-13myc than in cells bearing the GAL1 vector (Fig. 9G–I). And an accumulation of Clb2 could be observed in cells expressing Clb6ST-13myc at 30 min, cells expressing Clb6 at 60 min, and cells bearing the GAL1 vector at 90 min after release from G1 arrest (Fig. 9G–I). For some unknown reason, the protein level of Pds1-3HA went down faster in cells over-expressing Clb6ST13myc than in cells over-expressing Clb6-13myc starting approximately at 90 min after release from G1 arrest (Fig. 9G–I). Overall, these observations indicated that overexpression of Clb6ST-13myc could promote an early expression of Pds1 and Clb2.

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3.9. Overexpression of Clb6ST exacerbates the defect in budding in swe1D cells but still promotes S phase entry It is known that Clb2/Cdc28 kinase can prevent bud formation and initiates mitosis (Amon et al., 1994; Irniger et al., 1995; Padmashree and Surana, 2001; Surana et al., 1991). Here, our observations showed that an early expression of Clb2 resulting from overexpression of Clb6 or Clb6ST did not prevent budding nor promote the entry into mitosis (Fig. 9). Previous observations had shown that the enhanced expression of Cdc6, an inhibitor of Clb/Cdc28 complex, induced by overexpression of Clb6 can inhibit mitotic Clb/Cdc28 and prevent the entry into mitosis (Basco et al., 1995; Elsasser et al., 1996). Furthermore, it should be noted that Swe1 is an inhibitory kinase for mitotic Clb/Cdc28 activity and accumulates starting in late G1 and persists until mitosis (Booher et al., 1993; Lew, 2003). Therefore, we speculated that Swe1 may inactivate early accumulated Clb2/Cdc28 resulting from overexpression of Clb6 or Clb6ST in late G1, which in turn allowed budding to occur. To test our hypothesis, we determined whether deletion of SWE1 could prevent budding in cells expressing Clb6ST. As described in the legend to Fig. 10, cells were examined at the indicated times after release from G1 arrest into fresh galactose medium for continuous induction of Clb6ST. At 120 min after release from G1 arrest, 55% of swe1D cells bearing the GAL1 vector and 35% of swe1D cells bearing GAL1-clb6ST-13myc were budded whereas 70% of SWE1 cells bearing the GAL1 vector and 75% of SWE1 cells bearing GAL1-clb6ST-13myc (Fig. 10A). This indicated that swe1D cells were defective in budding and that this phenotype was exacerbated by overexpression of Clb6ST. At the same time point, about 26% of swe1D cells bearing the GAL1 vector were budded with two nuclei whereas about 18% of SWE1 cells bearing the GAL1 vector. This is consistent with the previous observations (Harvey and Kellogg, 2003) where swe1D cells bearing the GAL1 vector appeared to enter mitosis earlier than SWE1 cells bearing the GAL1 vector. To investigate if swe1D cells were also defective in the S-phase entry, flow cytometric analyses were performed. The histogram profiles of DNA contents reveals that at 60 min. 49% of SWE1 cells bearing the GAL1 vector and 57% of swe1D cells bearing the GAL1 vector were still in G1 whereas only 16% of SWE1 cells bearing the GAL1-clb6ST-13myc and 19% of swe1D cells bearing the GAL1clb6ST-13myc in G1 (Fig. 10B). These observations indicate that swe1D cells were defective in the S-phase entry and that overexpression of Clb6ST-13myc could enhance the phenotype in budding but still promoted S-phase entry in swe1D cells. Indeed, the fact that the swe1D cells overexpressing Clb6ST13myc failed to bud normally made it difficult to examine the effect of swe1D mutation on delay in G2 and nuclear separation caused by overexpression of Clb6ST-13myc. However, at 240 min after release from G1 arrest, only 13% of swe1D cells overexpressing Clb6ST-13myc had a DNA contents of 1C as judged by flow cytometric analysis (Fig. 10B), but 56% of them were still unbudded with a single nucleus and 6% of them unbudded with two separated nuclei (Fig. 10A and Supplementary Fig. S5). Furthermore, 15% of swe1D cells overexpressing Clb6ST-13myc were budded with a single nucleus. Therefore, consistent with previous observation (Basco et al., 1995) swe1D mutation did not relieve G2 delay caused by overexpression of Clb6ST.

4. Discussion 4.1. Clb6 proteolysis is regulated by both the APCCdh1 and SCFCdc4 complexes It is known that the SCFCdc4 ubiquitin ligase is required for Clb6 proteolysis (Jackson et al., 2006). In this study, we examined the

relationship between Clb6 and Cdc14, an activator for APCCdh1, and found that Clb6 accumulated in cdc14ts cultures but not in CDC14 cultures at the permissive temperature when overexpressed (Fig. 1). Subsequent observations demonstrated that Clb6 proteolysis required Cdc16, Cdc4 and Cdh1, but not Cdc20, in early G1 (Figs. 2 and 3 and Supplementary Fig. S1). Clb6 contains both a KEN box and D-box sequence in the N-terminus. Mutations in these two sequences enhanced Clb6 stability and Clb6 was also observed to be a weak APCCdh1 substrate in vitro (Figs. 3 and 4). These observations suggest that, in addition to SCFCdc4, APCCdh1 is contributes to Clb6 proteolysis. Persistent APCCdh1 activity also promoted Clb6 proteolysis in late G1 (Figs. 5 and 6). However, we found that Clb6 was highly stable in cdc4-1 cdh1D cells arrested in early G1 but still not very stable in cdc4-1 cdh1D cells arrested in late G1 at the non-permissive temperature when Clb6 was over expressed by the GAL1 promoter (Figs. 3 and 5). Clb6 was still degradable in cdc4-1 cdh1D cells at the non-permissive temperature even when expressed from the CLB6 promoter (Fig. 6 and Supplementary Fig. S2). This indicates that some other unknown mechanism(s) must also play a significant role in the proteolysis of Clb6 proteins independently of both SCFCdc4 and APCCdh1 complexes in late G1. Our conclusion differs from those reported by Jackson et al. that APC is not required for proteolysis of Clb6 (Jackson et al., 2006). This could be due to the differences in experimental designs for the measurements of Clb6 stability. We examined the role of APC in Clb6 stability specifically in G1 when APC is known to be active. By contrast, Jackson et al. did not synchronize cells in G1 before they examined the effect of apc2-4 mutation on Clb6 stability at the non-permissive temperature (Jackson et al., 2006). The apc24 mutation may make APC lose activity and cells may arrest in mitosis at the non-permissive temperature (Kramer et al., 1998). Because APCCdh1 is only active from late mitosis to late G1 (Huang et al., 2001; Zachariae et al., 1998), it is possible that SCFCdc4 and the mechanism independent of both SCFCdc4 and APCCdh1 complexes are dominant in counteracting the effect of apc2-4 mutation on Clb6 stability. If APC activity contributes in a meaningful way to Clb6 protein stability, one prediction is that sequence alignment analyses should reveal evidence in Clb6 orthologs from other Saccharomyces species for a conserved KEN box and D-box. The sequence alignments for N-terminal 100 amino acids of Clb6 orthologs from four Saccharomyces species (S. cerevisiae, S. bayanus, S. mikatae, and S. paradoxus) reveals that the KEN box is conserved, and that the Dbox is present in S. cerevisiae, but not in three other Clb6 orthologs (Supplementary Fig. S6). Conservation of the KEN box in these four Clb6 orthologs suggests that a requirement of APCCdh1 for Clb6 proteolysis may also be evolutionarily conserved among these four Saccharomyces species. 4.2. APCCdh1-dependent proteolysis and CDK phosphorylation of Clb6 at the G1/S transition Sic1, a CDK inhibitor, binds and inhibits both Clb5/Cdc28 and Clb6/Cdc28 to prevent the G1/S transition (Nugroho and Mendenhall, 1994; Schwob et al., 1994). Furthermore, Sic1 proteolysis is dependent upon SCFCdc4 complex and Cln1-3/Cdc28 phosphorylation and is required for the G1-S transition as judged by the observation that sic1 deletion can make cln1 cln2 cln3 triple mutant or cdc4-arrested cells enter S-phase and replicate DNA (Feldman et al., 1997; Knapp et al., 1996; Schneider et al., 1996; Skowyra et al., 1997). In this study, our observations reveal that Clb6am expression under the native CLB6 promoter can make cells bypass late G1 arrest caused by cdc4-1 but Clb6mkd expression, Clb6S147A expression or Clb6ST expressed under the CLB6 promoter do not cause this bypass (Fig. 8A). This suggests that unlike Sic1,

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Fig. 10. Effect of swe1D mutation on cell cycle progression when Clb6ST is overexpressed. Wild type or swe1D strains bearing either the GAL1 vector or GAL1-clb6ST-13myc constructs were used for analyses. The experiments were performed as described in Fig. 9 except that 100 mL of YEP + sucrose and YEP + galactose mediums were used. The arrows indicate that at 150 min after release from G1 arrest, a-factor was added to make cells arrest in G1 of the subsequent cell cycle. The observations of morphology and nuclei of cells (A), and for DNA content (B) were also performed as described in Fig. 9. Open circle (s) unbudded cells with one nucleus; closed circle (d) budded cells with one nucleus; open square (h) budded cells with two nuclei; closed square (j) chained cells; open diamond (e) unbudded cells with two nuclei or budded cells with three or more nuclei.

APCCdh1-dependent Clb6 proteolysis and CDK phosphorylation of Clb6 at residue S147 are both required in combination to prevent the G1/S transition of cdc4-1 cells. However, since Clb6ST cannot make cells bypass late G1 arrest caused by cdc4-1, this indicates that CDK phosphorylation of Clb6 at S6 and T39 confers Clb6 with the ability to promote the G1-S transition and this effect can be counteracted by CDK phosphorylation of Clb6 at S147. We also found that compared to Clb6, Clb6-3A causes an increase in the number of unbudded cells and suppresses formation of multiple buds in cdc4-1 cells but that the Clb6S147A allele cannot (Fig. 8B). It is know that Cln/Cdc28 activity induces formation of multiple buds and Sic1 binds to and inhibits Clb5-6/Cdc28 to prevent DNA replication in cdc4ts late G1-arrested cells at the nonpermissive temperature (Amon et al., 1994; Blondel and Mann, 1996; Schwob et al., 1994; Singer et al., 1984). Furthermore, flow cytometric analyses revealed that Clb6-3A failed to allow cells to bypass the late G1 arrest caused by cdc4-1 (Fig. 8A). We speculate that Clb6-3A may compete with Cln1–Cln3 to bind to Cdc28 but Clb6-3A/Cdc28 is still inhibited by Sic1, which in turn leads to a decrease in formation of multiple buds.

Previous observations revealed that CDK phosphorylation at Ser6, Thr39 and S147 regulates SCFCdc4-dependent Clb6 degradation (Jackson et al., 2006). Our observations further reveal that CDK phosphorylation at Ser6, Thr39 and S147 regulates Clb6 function (Fig. 8). Taken together, we conclude that APCCdh1 and SCFCdc4dependent Clb6 proteolysis and CDK phosphorylation of Clb6 are used in combination to prevent a premature G1/S transition. 4.3. Significance of SCFCdc4- and APCCdh1-dependent proteolysis of Clb6 In considering our observations that Clb6 proteolysis was regulated in part by both the APCCdh1 and the SCFCdc4 complex, Clb6 and the stabilized version of Clb6 (Clb6ST) were expressed under the GAL1 promoter rather than by the CLB6 promoter. Our observations revealed that in G1 arrest/release experiments that Clb6 or Clb6ST accelerate budding and entry into S-phase, promote an earlier expression of Pds1 and Clb2 and resulted in a delay in G2 when overexpressed (Fig. 9). Furthermore, overexpression of Clb6ST could result in an accumulation of more chained cells than that of Clb6 in wild type cells (Table 1).

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The G1 cyclins Cln1-2 are required for bud emergence at the G1/ S border (Bloom and Cross, 2007). Clb6 has a negative role in regulating expression of Cln2, but Clb6 can perform the function of Cln1-2 in bud emergence (Basco et al., 1995; Schwob and Nasmyth, 1993). This may explain our observation that overexpression of Clb6 or Clb6ST significantly accelerated budding (Fig. 9). In addition, overexpression of Clb6ST promoted S-phase entry more significantly than that of Clb6 (Fig. 9). Nevertheless, our observations differ from previous ones in that Clb6overexpressing cells could bud and progress into S-phase nearly as well as control cells (Basco et al., 1995). This discrepancy seems to be due to the fact that in our analyses expression of Clb6 or Clb6ST was induced for 30 min in early G1 before cells were released from G1 arrest into galactose medium whereas in the analyses performed by Basco et al., small G1 cells were elutriated and then directly released into the galactose medium (Basco et al., 1995). Therefore, our observations revealed that an early expression of Clb6 or Clb6ST in early G1 were able to accelerate the G1/S transition. We found that as compared to wild type cells, swe1D cells were partially defective in bud formation and this defective phenotype was greatly exacerbated by overexpression of Clb6ST (Fig. 10). This indicates that overexpression of Clb6ST promotes budding requiring Swe1. It is known that Swe1 is an inhibitor for Clb2/Cdc28 and that Clb2/Cdc28 can prevent bud emergence (Amon et al., 1994; Irniger et al., 1995; Lew, 2003; Padmashree and Surana, 2001; Surana et al., 1991). Therefore, the enhanced phenotype of swe1D cells in budding could be attributed to an early expression of Clb2 induced by overexpression of Clb6ST (Fig. 9). Taken together, our observations suggest that in late G1 Clb6 proteolysis is required to prevent early expression of Clb2 and that Swe1 inhibits the early accumulation of Clb2/Cdc28 for bud emergence. It has been reported that Swi4 regulates CLB2 expression for negative feedback control and Mbp1 regulates CLB2 expression in a repressive way (Bean et al., 2005). The transcription factor Swi6 interacts with Swi4 to form the SBF complex, and with Mbp1, to form MBF complex to negatively or positively regulate expression of G1- and S-phase-specific genes (Koch et al., 1993). At the G1/S border, Swi6 is phosphorylated at serine 160 by Clb6/Cdc28 and then exported to the cytoplasm, which in turn may lead to inactivation of both SBF and MBF complexes (Geymonat et al., 2004). Therefore, in Fig. 9, the early expression of Clb2 that was induced by overexpression of Clb6 or Clb6ST could be due to Clb6/Cdc28 or Clb6ST/Cdc28 phosphorylating Swi6, which in turn led to inactivation of SBF and MBF in G1. It is also noted that in cells overexpressing Clb6ST both Pds1 and Clb2 were expressed simultaneously (Fig. 9), indicating that overexpression of Clb6ST could interfere with the timing and order of expression for both Pds1 and Clb2. It has been shown that Cdc6 binds to and inhibits mitotic Clb/ Cdc28, has a negative effect on entry into mitosis when overexpressed and that its enhanced expression induced by overexpression of Clb6 contributes to the delay in G2 (Basco et al., 1995; Elsasser et al., 1996). It has also been shown that Cdc6 has an inhibitory effect on the APCCdc20 activity, prevents nuclear separation and that this inhibition can be relieved by deletion of the phosphatase gene CDC55, but not by deletion of SWE1 (Boronat and Campbell, 2007). Our observations also excluded the possibility that Swe1 was involved in the delay in G2 caused by overexpression of Clb6ST (Fig. 10). Taken together, Cdc6 may be the key regulator to keep cells in G2 and prevent entry into mitosis when Clb6 is overexpressed, a hypothesis that needs further testing. Clb6 has been shown to be similar to mammalian cyclin E in many respects (Jackson et al., 2006). Both Clb6 and cyclin E are expressed in a short period at the G1/S border, substrates of CDK and are degraded via the SCF ubiquitin–proteasome pathway in a

CDK-dependent manner (Jackson et al., 2006). It is known that overexpression of cyclin E can result in an increase in frequency of polyploidy (Spruck et al., 1999). Here, we found that overexpression of Clb6 induced formation of chained cells in cdc14-1 cell culture and that as compared to Clb6, overexpression of Clb6ST had a greater effect on formation of cellular chains in wild type cell culture (Fig. 1 and Table 1). Formation of cellular chains is due to either a cytokinesis defect or a cell separation defect at the time of cell division. Therefore, this observation suggests that Clb6 proteolysis is required to ensure proper progression of cell division. Of note, because Clb6 is normally not expressed in the M-G1 transition, overexpression of Clb6 or Clb6ST should result in an indirect effect on cell division. In summary, as compared to Clb6, overexpression of Clb6ST had a greater effect on promoting Clb2 expression and S-phase entry, resulted in a greater G2 delay and higher accumulation of chained cells. Our observations reveal the significance of both SCFCdc4 and APCCdh1-dependent proteolysis of Clb6 on the expression of both PDS1 and CLB2, the timing of the G1/S transition, the timing of the G2/M transition and on the completion of cell division. Our observations also revealed that Swe1 could inhibit the early accumulation of Clb2/Cdc28 and hence ensures the occurrence of bud emergence at the G1/S border. 5. Conclusion Our observations reveal the significance of both SCFCdc4 and APCCdh1-dependent proteolysis of Clb6 on the timing of the G1/S transition, on the expression of PDS1 and CLB2, on the timing of the G2/M transition and on the completion of cell division. We have also revealed that Swe1 can inhibit the early accumulation of Clb2/Cdc28 and hence ensures the occurrence of bud emergence at the G1/S border. These data add to the concept that not only do properly executed mitotic exit events prepare cells to initiate the next round of DNA replication upon entry into S phase, but also that the proper execution of cell cycle regulated events at the end of G1 and at the entry into S phase are necessary for the proper execution of mitosis and mitotic exit. Acknowledgments We thank A. Amon, J.N. Huang, and D. Pellman for strains and/or plasmids. SCS was supported by the Chang Gung Memorial Hospital, the Taiwan Ministry of Science and Technology and the Ministry of Education (CMRPD1C0613, CMRPD1E0171 and BMRPC59, MOST103-2311-B-182-005-MY3, EMRPD1E1541 and EMRPD1F0161). YLJ was supported by Tzu-Chi University and Mackay Medical College (1021B02, 1031B01 and 1041B04). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2016.03.004. References Adams, A., Gottschling, D.E., Kaiser, C.A., Stearns, T., 1997. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Plainview, New York. Amon, A., Irniger, S., Nasmyth, K., 1994. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell 77, 1037–1050. Basco, R.D., Segal, M.D., Reed, S.I., 1995. Negative regulation of G1 and G2 by Sphase cyclins of Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 5030–5042. Bean, J.M., Siggia, E.D., Cross, F.R., 2005. High functional overlap between MluI cellcycle box binding factor and Swi4/6 cell-cycle box binding factor in the G1/S transcriptional program in Saccharomyces cerevisiae. Genetics 171, 49–61.

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