Materials and Methods: All strains were derivatives of SK1 and are listed in Supplemental Table 1.

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1 Supplemental Online Material: Materials and Methods: All strains were derivatives of SK1 and are listed in Supplemental Table 1. Constructs: pclb2-3ha-cdc5 and pclb2-3ha-cdc2 strains were constructed by a one step PCR based gene replacement method (S1) with the pfa6a-pclb2-3ha- KanMX6 plasmid as template. The pfa6a-pclb2-3ha-kanmx6 plasmid was constructed by replacing the GAL1 promoter fragment in pfa6a-gal-3ha-kanmx6 (S1) with a 1kb PCR fragment of the CLB2 promoter. The pscc1-3ha-pds1 strains were constructed by PCR replacing the PDS1 promoter with the SCC1 promoter with pfa6a-pscc1-3ha-kanmx6 as template. The pfa6a-pscc1-3ha-kanmx6 plasmid was constructed as described above except that a 1kb PCR fragment containing the SCC1 promoter was used instead of the CLB2 promoter. REC8-3HA and URA-GFP dots were described in (S2). NDC1-13MYC, UBR1 and MAM1 deletions were constructed by a one-step PCR based gene replacement method (S1). MAM1-9MYC, NDC1-6HA, prec8-scc1-3ha, SPO11 and REC8 deletion as well as the CEN-GFP dots were described in (S3). PDS1-18MYC was described in (S4) 3HA-CDC14, the SPO12 deletion construct and the temperature sensitive cdc14-1 mutant were described in (S5). Sporulation condition: Cells were grown to saturation in YPD (YEP + 2% glucose) for 24 hours, diluted into YPA (YEP + 2% KAc) at OD 6 =.25 and grown over night. The cells were then washed with water and resuspended in SPO medium (.3% KAc, ph = 7.) at OD 6 = 1.85 to induce sporulation. Cells were grown at 3 C unless otherwise indicated. CIP phosphatase treatment: Extracts were prepared as described in (S6). Extracts were split in half and subjected to immunoprecipitation using appropriate antibodies. Imunoprecipitates were incubated with either buffer alone or buffer containing 8 units of Calf Intestinal Alkaline Phosphatase (CIP) at 37 C for 3min and were then analyzed by Western blot analysis.

2 Immunofluorescence techniques: Indirect in situ immunofluorescence was carried out as described in (S7). Rat -tubulin antibodies (Oxford Biotechnology) were use at a 1:1 dilution and -rat FITC or -rat Rhodamine antibodies (Jackson ImmunoResearch) at a 1:1 dilution. To detect Mam1-9MYC, mouse -MYC antibodies (Babco) were used at a 1:25 and -mouse FITC antibodies (Jackson ImmunoResearch) at a 1:1 dilution. 3HA-Cdc14 was detected with mouse -HA antibody (Covance) at a1:5 dilution and mouse CY3 (Jackson ImmunoResearch) at 1:5 dilution. Metaphase I cells were defined as cells with one DNA mass spanned by a short thick meiotic spindle measuring 2 3 µm in length. Anaphase I cells were defined as cells with spindles measuring at least 5 µm. Metaphase II and anaphase II cells were defined in a similar manner. Binucleate cells were defined as cells with two distinct but not necessarily separated DNA masses. Tetranucleate cells were defined in a similar manner. GFP, green fluorescence protein, chromosome dots: Heterozygous URA3 GFP dot: An array of TET operator sites was integrated at the URA3 locus, 35kb from the centromere of one chromosome V (S2). A TET repressor GFP fusion was also express in this strain to visualized the location of the of TET operator sites. Heterozygous CEN GFP dots were constructed in a similar manner except the array of TET operator sites were integrated 1.4kb from the centromere of one homolog of chromosome V (S3). Meiotic Spreads: Chromosomes were spread as described by (S8). Rec8-3HA was detected using an -HA antibody at a 1:5 dilution and an -mouse CY3 antibody at a 1:1 dilution. Ndc1-13MYC was detected using a rabbit -MYC antibody (Gramsch) at a 1:2 dilution and an -rabbit FITC antibody (Jackson ImmunoResearch) at 1:3 dilution. Mam1-9MYC was detected using a rabbit anti-myc antibody at a 1:6 and an -rabbit FITC antibody at a 1:1 dilution. Ndc1-6HA was detected using a mouse -HA antibody at a 1:32 and an -mouse CY3 antibody at a 1:3 dilution.

3 Quantification of Mam1 localization: Mam1 was determined to localize to kinetochores when more than 6 percent of Mam1 dots co-localized with Ndc1 dots on chromosome spreads. Note to the readers: Throughout this manuscript we define cell cycle stages by meiotic spindle morphology. Using this definition both, Cdc2- and Cdc5-depleted cells arrest in metaphase I. It is, however, important to note that the metaphase I arrest exhibited by Cdc2-depleted cells differs from that of Cdc5-depleted cells at the molecular level. In Cdc2-depleted cells Pds1 is not degraded whereas the protein is eventually degraded in Cdc5-depleted cells. Furthermore, sister kinetochores are co-oriented in Cdc2-depleted cells but bi-oriented in Cdc5-depleted cells.

4 A Time (Hrs) Pds1-18Myc WT pclb2-cdc pclb2-cdc Rec8-3HA Pgk3 B % Cells spo11, pscc1-3ha-pds1, pclb2-3ha-cdc5 MI spo11, pscc1-3ha-pds1, pclb2-3ha-cdc5 MII spo11, pscc1-3ha-pds1 MI spo11, pscc1-3ha-pds1 MII spo11 MI spo11 MII % Metaphase I Spindles spo11, pscc1-3ha-pds1, pclb2-3ha-cdc5 spo11, pscc1-3ha-pds1 spo Time in Hrs Time in Hrs Time in Hrs % Anasphase I to Anaphase II Spindles spo11, pscc1-3ha-pds1, pclb2-3ha-cdc5 spo11, pscc1-3ha-pds1 spo11 Supplemental Figure 1

5 Figure S1: Pds1 is stabilized in Cdc5-depleted cells but Pds1 stabilization is not the sole reason for the metaphase I arrest exhibited by the mutant. (A) Wild type (A8188), pclb2-cdc5 (A8189) and pclb2-cdc2 (A819) carrying a PDS1-18MYC fusion and a REC8-3HA fusion were sporulated as described in Materials and Methods. Pds1-18MYC, Rec8-3HA, and Pgk3 (loading control) were monitored by Western blot analysis (S6). (B) spo11 (A2983), spo11 pscc1-3ha-pds1 (A7859) and spo11 pscc1-3ha- PDS1 pclb2-3ha-cdc5 (A785) were sporulated as described in Materials and Methods. The percentage of cells with two or more nuclei (closed symbols, MI) three or four nuclei (open symbols, MII, left graph) metaphase I spindles (middle graph) and the sum of anaphase I and meiosis II spindles (right graph) was determined. Pds1 produced from the Scc1 promoter is absent during meiosis as judged by Western blot analysis (S9). spo11 cells do not accumulate metaphase I spindles as the formation of stable metaphase I spindles requires tension provided by chiasmata which are absent in spo11 cells. Instead these cells elongate their spindle and enter anaphase I prematurely (S4). The fact that at least a fraction of Cdc5-depleted cells lacking PDS1 arrest with metaphase I spindles indicates that some Rec8 is retained on chromosomes. Thus, Cdc5 appears to be important not only for Pds1 degradation but also for Rec8 removal from chromosomes after Pds1 inactivation. During mitosis, Cdc5 s role in aiding in the cleavage of Scc1/Mcd1 is minor (S1). Our results suggest that the protein kinase is more important if not critical for this process during meiosis.

6 pclb2-cdc2 pclb2-cdc5 Wild type no cohesin Wild type Cen only Wild type Full Length Rec8-3HA Ndc1-13MYC DAPI Merged Supplemental Figure 2

7 Figure S2: Localization of Rec8 on chromosomes. Examples of Rec8 localization of strains described in Figure 2C. Rec8 localized along the entire length of the chromosome (full length localization) in wild-type cells (top panel), Rec8 localized to centromeric regions (Cen only) of anaphase I wild-type cells (second panel). No Rec8 staining is detected in wild-type anaphase II cells (no cohesion) in wild-type cells (third panel). Rec8 localization in pclb2-cdc5 and pclb2-cdc2 cells, respectively (forth and bottom panel). Rec8-3HA is shown in red, Ndc1-13MYC in green and DAPI in blue.

8 A % Metaphase I Spindles spo11 pclb2-3ha-cdc2 spo11 pclb2-3ha-cdc5 spo11 % Anaphase I Spindles % Metaphase II Spindles % Anaphase II Spindles Time in Hrs Time in Hrs Time in Hrs Time in Hrs B % Cells spo11, rec8, pclb2-3ha-cdc5 MI 7 spo11 rec8 pclb2-3ha-cdc5 spo11, rec8, pclb2-3ha-cdc5 MII spo11 pclb2-3ha-cdc5 spo11, pclb2-3ha-cdc5 MI 6 spo11 rec8 spo11, pclb2-3ha-cdc5 MII spo11, rec8 MI 5 spo11, rec8 MII % Metaphase I Spindles % Anaphase I Spindles % Meiosis II Spindles Time in Hrs Time in Hrs Time in Hrs Time in Hrs Supplemental Figure 3

9 Figure S3: Preventing recombination allows pclb2-cdc2 cells to enter anaphase I, whereas loss of REC8 allows anaphase I entry in CDC5-depleted cells. (A) spo11 (, A2983), spo11 pclb2-3ha-cdc5 (, A5764) and spo11 pclb2-3ha-cdc2 (, A5459) were sporulated as described in Materials and Methods. The percentage of cells with metaphase I spindles (first graph), anaphase I spindles (second graph), metaphase II spindles (third graph) and anaphase II spindles (fourth graph) was determined. spo11 cells do not accumulate metaphase I spindles as the formation of stable metaphase I spindles requires tension provided by chiasmata which is absent in spo11 cells. Instead these cells elongate their spindle and enter anaphase I prematurely (S4). (B) spo11 rec8 pclb2-3ha-cdc5 (A5457), spo11 pclb2-3ha-cdc5 (A5764) and spo11 rec8 (A389) were sporulated as described in Materials and Methods. The percentage of cells with two or more nuclei (closed symbols, MI, first graph) three or four nuclei (open symbols, MII, first graph) metaphase I spindles (C, second graph), anaphase I spindles (C, third graph) and meiosis II spindles (C, last graph) was determined.

10 Percentage of Anaphase I Cells Cdc14 Release from the Nucleolus full partial control pclb2-cdc5 Supplemental Figure 4

11 Figure S4: CDC5 is required for Cdc14 release from the nucleolus during anaphase I. spo11 rec8 (A8175) and pclb2-cdc5 spo11 rec8 ( 8157) containing a heterozygous 3HA-CDC14 fusion were sporulated as described in Materials and Methods. Cdc14 localization was determined in 15 anaphase I cells. Partial release was defined as 3HA-Cdc14 staining present in the nucleus but with nucleolar enrichment. Full released was defined as an even staining of 3HA-Cdc14 throughout the nucleus. CDC5 is required for exit from mitosis through its role in promoting the release of the protein phosphatase Cdc14 from the nucleolus during anaphase (reviewed in (S11). These results indicate that CDC5 is also essential for this process during anaphase I.

A pclb2-cdc2 pclb2-3ha-cdc5 pclb2-3ha-cdc5 CENV dot

Spindles 8 6 4 2 1 2 3 4 5 6 7 8 9 Time (Hrs) %

pclb2-3ha-cdc5 pclb2-cdc2 mam1 pclb2-cdc2

pclb2-cdc2 spo12 mam1 1 2 3 4 5 6 7 8 9 Time (Hrs) %

12 A pclb2-cdc2 pclb2-3ha-cdc5 pclb2-3ha-cdc5 CENV dot DAPI CENV dot DAPI CENV dot DAPI B % Metaphase I Spindles Time (Hrs) % Anaphase I to Anaphase II Spindles pclb2-cdc2 pclb2-3ha-cdc5 pclb2-cdc2 mam1 pclb2-cdc2 pclb2-3ha-cdc5 pclb2-cdc2 spo12 pclb2-cdc2 cdc14-1 pclb2-cdc2 spo12 mam Time (Hrs) % CENV GFP Separation Time (Hrs) Supplemental Figure 5

13 Figure S5: Cdc5-depleted cells but not FEAR network mutants are defective in sister kinetochore co-orientation during metaphase I. (A) Examples of centromere separation in Cdc5-depleted metaphase I cells. The left panel shows the behavior of co-oriented sister kinetochores during metaphase I in Cdc2- depleted cells carrying a CEN-GFP dot 1.4kb from the centromere of one homolog of chromosome V (heterozygous CEN-GFP dot). The middle and right panels show examples of sister kinetochore separation observed in Cdc5-depleted metaphase I cells carrying heterozygous CEN-GFP dots, which is indicative of sister kinetochores being bioriented. Genotypes of strains are described in Figure 3. CEN-GFP dots are shown in green, DNA in blue. Pictures of cells were taken 8 hours after the induction of sporulation. (B) pclb2-cdc2 (, A7118), pclb2-3ha-cdc5 (, A7151), pclb2-cdc2 mam1 (, A7316), pclb2-cdc2 pclb2-3ha-cdc5 (, A8328), pclb2-cdc2 spo12 (, A8326), pclb2-cdc2 cdc14-1 (, A8369) and pclb2-cdc2 spo12 mam1 (, A8327) containing heterozygous CEN-GFP dot were sporulated as described in Materials and Methods, except that cells were grown at room temperature in YPD and YPA and were then shifted to 3 C in SPO medium to inactivate cdc14-1. The percentage of cells with metaphase I spindles (left panel), sum of anaphase I and meiosis II spindles (middle panel) and separated CEN-GFP dots (right panel) was determined. Cdc5 is a component of the FEAR network, which is required to promote the release of the protein phosphatase Cdc14 from the nucleolus during anaphase (S12). Cdc14 released from the nucleolus then promotes exit from mitosis (reviewed in (S11)). During meiosis, inactivation of the FEAR network or CDC14 leads to an uncoupling of meiotic events. Cells carrying a FEAR network or CDC14 mutation initiate many aspects of meiosis II despite defects in anaphase I spindle disassembly (S5, 13). It was, therefore, possible that Cdc5-depleted cells exhibited this peculiar uncoupling phenotype and that sister kinetochore bi-orientation was not occurring during meiosis I but when meiosis II was scheduled to occur. To address this possibility we compared the behavior of sister kinetochores of Cdc5-depleted cells with that of another FEAR network mutant (spo12 )

14 and with that of a cdc14-1 mutant under two experimental conditions. In the first experiment we compared the segregation pattern of chromosome V of Cdc5-depleted cells with that of cells lacking SPO12 that had the REC8 open reading frame replaced by SCC1/MCD1. In Cdc5-depleted cells sister chromatids segregated from each other, whereas in spo12 cells homologs disjoined (Figure 3B). In the second experiment we compared the kinetics of centromere separation of Cdc5-depleted cells with that of cells lacking SPO12 or cells carrying a cdc14-1 mutation. As cdc14-1 mutants and spo12 mutants do not arrest in metaphase I, we conducted this experiment in cells depleted for Cdc2. Cdc5- Cdc2- double depleted cells separated sister kinetochores in metaphase I, with kinetics similar to that of pclb-cdc2 mam1 cells. In contrast, Cdc2-depleted cells carrying a cdc14-1 mutation or a SPO12 deletion did not separate sister kinetochores. This was not due to a failure to detect sister centromere separation in these cells as pclb2-cdc2 mam1 spo12 cells did separate sister centromeres. Together, these findings indicate that sister kinetochore bi-orientation in the metaphase I arrest of Cdc5-depleted cells is not a FEAR network mutant phenotype. The observation that sister kinetochores are bi-oriented even in Cdc5- Cdc2- doubly depleted cells further suggests that sister kinetochores are bi-oriented even under conditions when Pds1 degradation is prevented.

15 pclb2-cdc5 Wild type pclb2-cdc5 pclb2-cdc5 + CIP Wild type Wild type + CIP A Time (Hrs) Mam1-9MYC Pgk3 B Mam1-9MYC Wild type pclb2-cdc5 pclb2-cdc C Mam1-9MYC Supplemental Figure 6

16 Figure S6: CDC5 is required for Mam1 phosphorylation. (A) Wild type (A797), pclb2-cdc5 (A7449) and pclb2-cdc2 (A745) carrying a MAM1-9MYC and NDC1-6HA fusions were sporulated as described in Materials and Methods. Pgk3 and Mam1 protein level were monitored by Western blot analysis. (B) Analysis of Mam1 mobility in SDS PAGE in Cdc5-depleted (A7449) and wild-type (A797) cells 5hrs after the induction of sporulation. (C) Wild-type (A797) and Cdc5-depleted cells (A7449) cells were sporulated as described in Materials and Methods and samples were collected 5 hrs after the induction of sporulation. Mam1 was immunopreciptiated and either incubated with buffer or 8 units of Calf Intestinal Alkaline Phosphatase as described as in Materials and Methods.

17 Table S1: Strain List Strain number A512 A5128 A4964 A5844 A5823 A3685 A274 A6143 A6144 A4758 A6136 A6586 A6245 A6321 A6342 A8279 A7118 A7151 A7316 A7117 A5762 A746 A797 A7449 Relevant genotype MATa/ pclb2-3ha-cdc5/pclb2-3ha-cdc5 MATa/ pclb2-3ha-cdc2/pclb2-3ha-cdc2 MATa/ PDS1-18MYC/+ REC8-3HA/+ MATa/ PDS1-18MYC/+ REC8-3HA/+ pclb2-cdc5/pclb2-cdc5 MATa/ PDS1-18MYC/+ REC8-3HA/+ pclb2-cdc2/pclb2-cdc2 MATa/ PDS1-18MYC/+ MATa/ ubr1 /ubr1 REC8-3HA/REC8-3HA MATa/ ubr1 /ubr1 REC8-3HA/REC8-3HA pclb2-cdc5/pclb2- CDC5 MATa/ ubr1 /ubr1 REC8-3HA/REC8-3HA pclb2-cdc2/pclb2- CDC2 MATa/ NDC1-13MYC/+ REC8-3HA/+ MATa/ NDC1-13MYC/+ REC8-3HA/+ pclb2-cdc5/pclb2-cdc5 MATa/ NDC1-13MYC/+ REC8-3HA/+ pclb2-cdc2/pclb2-cdc2 MATa/ spo11 /spo11 rec8 /rec8 prec8::prec8-scc1-3ha// prec8::prec8-scc1-3ha LEU2::pURA3-TetR-GFP /+ URA3::TetOx2243/+ MATa/ spo11 /spo11 rec8 /rec8 prec8::prec8-scc1-3ha// prec8::prec8-scc1-3ha LEU2::pURA3-TetR-GFP /+ URA3::TetOx2243/+ pclb2-cdc2/pclb2-cdc2 MATa/ spo11 /spo11 rec8 /rec8 prec8::prec8-scc1-3ha// prec8::prec8-scc1-3ha LEU2::pURA3-TetR-GFP /+ URA3::TetOx2243/+ pclb2-cdc5/pclb2-cdc5 MATa/ spo11 /spo11 rec8 /rec8 prec8::prec8-scc1-3ha// prec8::prec8-scc1-3ha LEU2::pURA3-TetR-GFP /+ URA3::TetOx2243/+ spo12 /spo12 MATa/ LEU2::pURA3-TetR-GFP /+ CENV::TetOx2243/+ pclb2- CDC2/pCLB2-CDC2 MATa/ LEU2::pURA3-TetR-GFP /+ CENV::TetOx2243/+ pclb2-cdc- 3HA-CDC5/pCLB2-3HA-CDC5 MATa/ LEU2::pURA3-TetR-GFP /+ CENV::TetOx2243/+ pclb2- CDC2/pCLB2-CDC2 mam1 /mam1 MATa/ LEU2::pURA3-TetR-GFP /+ URA3::TetOx2243/+ pclb2- CDC2/pCLB2-CDC2 MATa/ LEU2::pURA3-TetR-GFP /+ URA3::TetOx2243/+ pclb2-cdc- 3HA-CDC5/pCLB2-3HA-CDC5 MATa/ LEU2::pURA3-TetR-GFP /+ URA3::TetOx2243/+ pclb2- CDC2/pCLB2-CDC2 mam1 /mam1 MATa/ MAM1-9MYC/MAM1-9MYC NDC1-6HA/NDC1-6HA MATa/ MAM1-9MYC/MAM1-9MYC NDC1-6HA/NDC1-6HA pclb2-

18 A745 A2983 A7859 A785 A8188 A8189 A819 A5764 A5459 A5457 A389 A8175 A8157 A8328 A8326 A8369 A8327 CDC5/pCLB2-CDC5 MATa/ MAM1-9MYC/MAM1-9MYC NDC1-6HA/NDC1-6HA pclb2- CDC2/pCLB2-CDC2 MATa/ spo11 /spo11 MATa/ spo11 /spo11 pscc1-3ha-pds1/ pscc1-3ha-pds1 MATa/ spo11 /spo11 pscc1-3ha-pds1/ pscc1-3ha-pds1 pclb2- CDC-3HA-CDC5/pCLB2-3HA-CDC5 MATa/ PDS1-18MYC/ PDS1-18MYC REC8-3HA/+ MATa/ PDS1-18MYC/ PDS1-18MYC REC8-3HA/+ pclb2- CDC5/pCLB2-CDC5 MATa/ PDS1-18MYC/ PDS1-18MYC REC8-3HA/+ pclb2- CDC2/pCLB2-CDC2 MATa/ spo11 /spo11 pclb2-3ha-cdc5/pclb2-3ha-cdc5 MATa/ spo11 /spo11 pclb2-3ha-cdc2/pclb2-3ha-cdc2 MATa/ spo11 /spo11 rec8 /rec8 pclb2-3ha-cdc5/pclb2-3ha- CDC5 MATa/ spo11 /spo11 rec8 /rec8 MATa/ spo11 /spo11 rec8 /rec8 3HA-CDC14/3HA-CDC14 MATa/ spo11 /spo11 rec8 /rec8 3HA-CDC14/3HA-CDC14 pclb2- CDC5/pCLB2-CDC5 MATa/ LEU2::pURA3-TetR-GFP /+ CENV::TetOx2243/+ pclb2- CDC2/pCLB2-CDC2 pclb2-cdc-3ha-cdc5/pclb2-3ha-cdc5 MATa/ LEU2::pURA3-TetR-GFP /+ CENV::TetOx2243/+ pclb2- CDC2/pCLB2-CDC2 spo12 /spo12 MATa/ LEU2::pURA3-TetR-GFP /+ CENV::TetOx2243/+ pclb2- CDC2/pCLB2-CDC2 cdc14 /cdc14 TRP1::cdc14-1/ TRP1::cdc14-1 MATa/ LEU2::pURA3-TetR-GFP /+ CENV::TetOx2243/+ pclb2- CDC2/pCLB2-CDC2 mam1 /mam1 spo12 /spo12 S1. M. S. Longtine et al., Yeast 14, 953 (1998). S2. F. Klein et al., Cell 98, 91 (1999). S3. A. Toth et al., Cell 13, 1155 (2). S4. M. A. Shonn, R. McCarroll, A. W. Murray, Science 289, 3 (2). S5. A. Marston, B. Lee, A. Amon, submitted. S6. T. Moll, G. Tebb, U. Surana, H. Robitsch, K. Nasmyth, Cell 66, 743 (1991). S7. R. Visintin et al., Mol Cell 2, 79 (1998). S8. K. Nairz, F. Klein, Genes Dev 11, 2272 (1997). S9. B. Lee, unpublished observations. S1. G. Alexandru, F. Uhlmann, K. Mechtler, M. A. Poupart, K. Nasmyth, Cell 15, 459 (21). S11. A. J. Bardin, A. Amon, Nat Rev Mol Cell Biol 2, 815 (21).

19 S12. F. Stegmeier, R. Visintin, A. Amon, Cell 18, 27 (22). S13. S. B. Buonomo et al., submitted.

Figure S1, related to Figure 1. Characterization of biosensor behavior in vivo.

Figure S1, related to Figure 1. Characterization of biosensor behavior in vivo. Developmental Cell, Volume 23 Supplemental Information Separase Biosensor Reveals that Cohesin Cleavage Timing Depends on Phosphatase PP2A Cdc55 Regulation Gilad Yaakov, Kurt Thorn, and David O. Morgan