Rec8 Phosphorylation by Casein Kinase 1 and Cdc7-Dbf4 Kinase Regulates Cohesin Cleavage by Separase during Meiosis

During meiosis, two rounds of chromosome segregation after a single round of DNA replication produce haploid gametes from diploid precursors. At meiosis I, maternal and paternal kinetochores are pulled toward opposite poles, and chiasmata holding bivalent chromosomes together are resolved by cleavage of cohesin's α-kleisin subunit (Rec8) along chromosome arms. This creates dyad chromosomes containing a pair of chromatids joined solely by cohesin at centromeres that had resisted cleavage. The discovery that centromeric Rec8 is protected from separase during meiosis I by shugoshin/MEI-S332 proteins that bind PP2A phosphatase suggests that phosphorylation either of separase or cohesin may be necessary for Rec8 cleavage. We show here that multiple phosphorylation sites within Rec8 as well as two different kinases, casein kinase 1δ/ɛ (CK1δ/ɛ) and Dbf4-dependent Cdc7 kinase (DDK), are required for Rec8 cleavage and meiosis I nuclear division. Rec8 with phosphomimetic mutations is no longer protected from separase at centromeres and is cleaved even when the two kinases are inhibited. Our data suggest that PP2A protects centromeric cohesion by opposing CK1δ/ɛ- and DDK-dependent phosphorylation of Rec8. During meiosis, two rounds of chromosome segregation after a single round of DNA replication produce haploid gametes from diploid precursors. At meiosis I, maternal and paternal kinetochores are pulled toward opposite poles, and chiasmata holding bivalent chromosomes together are resolved by cleavage of cohesin's α-kleisin subunit (Rec8) along chromosome arms. This creates dyad chromosomes containing a pair of chromatids joined solely by cohesin at centromeres that had resisted cleavage. The discovery that centromeric Rec8 is protected from separase during meiosis I by shugoshin/MEI-S332 proteins that bind PP2A phosphatase suggests that phosphorylation either of separase or cohesin may be necessary for Rec8 cleavage. We show here that multiple phosphorylation sites within Rec8 as well as two different kinases, casein kinase 1δ/ɛ (CK1δ/ɛ) and Dbf4-dependent Cdc7 kinase (DDK), are required for Rec8 cleavage and meiosis I nuclear division. Rec8 with phosphomimetic mutations is no longer protected from separase at centromeres and is cleaved even when the two kinases are inhibited. Our data suggest that PP2A protects centromeric cohesion by opposing CK1δ/ɛ- and DDK-dependent phosphorylation of Rec8. Cleavage of cohesin at meiosis I requires phosphorylation of its Rec8 subunit Nonphosphorylatable Rec8 blocks the resolution of chiasmata in a dominant manner Separase preferentially cleaves Rec8 phosphorylated by the kinases DDK and CK1 PP2A protects centromeric cohesin at meiosis I by local dephosphorylation of Rec8 During mitosis, a multisubunit complex called cohesin entraps sister chromatids within a large proteinaceous ring and thereby holds them together from their creation during S phase until their disjunction to opposite halves of the cell at anaphase. By resisting the tendency of microtubules to pull sister chromatids apart during metaphase, cohesin creates the tension thought to be necessary to stabilize selectively amphitelic microtubule-kinetochore attachments, which connect sister chromatids to opposite spindle poles. Sister chromatids are eventually disjoined by separase, a thiol protease that cleaves cohesin's α-kleisin subunit Scc1/Rad21, opens cohesin's tripartite ring, and releases sister chromatids from their embrace. Separase is kept inactive for most of the cell cycle by the binding of an inhibitory chaperone known as securin (Pds1 in yeast), whose destruction at the hands of a ubiquitin ligase called the anaphase-promoting complex (APC/C) only takes place after all chromosomes have bioriented, i.e., attached to microtubules in an amphitelic manner. During meiosis, two rounds of chromosome segregation (meiosis I and II) without an intervening round of DNA replication produce haploid gametes from diploid progenitors. This is made possible by the lack of DNA replication between the meiotic divisions and by three key features of meiosis I (Petronczki et al., 2003Petronczki M. Siomos M.F. Nasmyth K. Un ménage à quatre: the molecular biology of chromosome segregation in meiosis.Cell. 2003; 112: 423-440Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). First, reciprocal recombination between homologous nonsister chromatids produces chiasmata that hold all four homologous chromatids together, thereby forming bivalent chromosomes. Second, monopolin proteins prevent the traction of sister kinetochores toward opposite spindle poles. As a consequence, the tension necessary to stabilize microtubule-kinetochore interactions is generated by pulling maternal and paternal sister kinetochore pairs (and not sisters) in opposite directions. Third, separase cleaves cohesin along chromosome arms, but not at centromeres. This resolves chiasmata and triggers the disjunction to opposite poles of dyad chromosomes containing a pair of chromatids joined solely at their centromeres by cohesin that had resisted cleavage. The latter is essential for the subsequent amphitelic attachment of sister kinetochores during meiosis II and is eventually destroyed by a second round of separase activity. The trigger for both meiotic divisions is thought to be identical to that during mitosis, namely, the ubiquitinylation of securin (and cyclin B) by the APC/C, producing a burst of separase activity that cleaves cohesin's meiosis-specific α-kleisin subunit Rec8 (Buonomo et al., 2000Buonomo S.B. Clyne R.K. Fuchs J. Loidl J. Uhlmann F. Nasmyth K. Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.Cell. 2000; 103: 387-398Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, Kitajima et al., 2003Kitajima T.S. Miyazaki Y. Yamamoto M. Watanabe Y. Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast.EMBO J. 2003; 22: 5643-5653Crossref PubMed Scopus (109) Google Scholar, Kudo et al., 2006Kudo N.R. Wassmann K. Anger M. Schuh M. Wirth K.G. Xu H. Helmhart W. Kudo H. McKay M. Maro B. et al.Resolution of chiasmata in oocytes requires separase-mediated proteolysis.Cell. 2006; 126: 135-146Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The persistence of cohesin at centromeres after the first meiotic division explains the unusual ability of meiotic cells to undergo a second round of chromosome segregation (meiosis II) without a preceding round of DNA replication during which cohesion is normally established (Nasmyth and Haering, 2005Nasmyth K. Haering C.H. The structure and function of SMC and kleisin complexes.Annu. Rev. Biochem. 2005; 74: 595-648Crossref PubMed Scopus (500) Google Scholar). What determines the different fates of cohesin at centromeres and on chromosome arms after the first wave of separase activity? Recent work has established that orthologs of the Drosophila MEI-S332 protein, called shugoshins, are required (Katis et al., 2004aKatis V.L. Galova M. Rabitsch K.P. Gregan J. Nasmyth K. Maintenance of cohesin at centromeres after meiosis I in budding yeast requires a kinetochore-associated protein related to MEI-S332.Curr. Biol. 2004; 14: 560-572Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, Kerrebrock et al., 1995Kerrebrock A.W. Moore D.P. Wu J.S. Orr-Weaver T.L. Mei-S332, a Drosophila protein required for sister-chromatid cohesion, can localize to meiotic centromere regions.Cell. 1995; 83: 247-256Abstract Full Text PDF PubMed Scopus (220) Google Scholar, Kitajima et al., 2004Kitajima T.S. Kawashima S.A. Watanabe Y. The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis.Nature. 2004; 427: 510-517Crossref PubMed Scopus (467) Google Scholar, Marston et al., 2004Marston A.L. Tham W.H. Shah H. Amon A. A genome-wide screen identifies genes required for centromeric cohesion.Science. 2004; 303: 1367-1370Crossref PubMed Scopus (228) Google Scholar, Rabitsch et al., 2004Rabitsch K.P. Gregan J. Schleiffer A. Javerzat J.P. Eisenhaber F. Nasmyth K. Two fission yeast homologs of Drosophila Mei-S332 are required for chromosome segregation during meiosis I and II.Curr. Biol. 2004; 14: 287-301Abstract Full Text Full Text PDF PubMed Google Scholar). Shugoshins are thought to control Rec8 cleavage by recruiting to kinetochores a PP2A phosphatase complex with a regulatory subunit of the B′ type (Rts1 in yeast) (Kitajima et al., 2006Kitajima T.S. Sakuno T. Ishiguro K. Iemura S. Natsume T. Kawashima S.A. Watanabe Y. Shugoshin collaborates with protein phosphatase 2A to protect cohesin.Nature. 2006; 441: 46-52Crossref PubMed Scopus (463) Google Scholar, Riedel et al., 2006Riedel C.G. Katis V.L. Katou Y. Mori S. Itoh T. Helmhart W. Gálová M. Petronczki M. Gregan J. Cetin B. et al.Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I.Nature. 2006; 441: 53-61Crossref PubMed Scopus (366) Google Scholar). Crucially, a budding yeast shugoshin mutant defective solely in the binding to PP2A fails to protect centromeric Rec8 in meiosis I (Xu et al., 2009Xu Z. Cetin B. Anger M. Cho U.S. Helmhart W. Nasmyth K. Xu W. Structure and function of the PP2A-shugoshin interaction.Mol. Cell. 2009; 35: 426-441Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The finding that shugoshins protect centromeric cohesin by recruiting PP2A implies that the phosphorylation of some protein is necessary for Rec8 cleavage. Candidates include Rec8 itself and separase. A clue that Rec8 might be PP2A's target is the finding that yeast cells expressing Scc1 instead of Rec8 during meiosis fail to protect centromeric cohesin, at least when recombination has been eliminated (Tóth et al., 2000Tóth A. Rabitsch K.P. Gálová M. Schleiffer A. Buonomo S.B. Nasmyth K. Functional genomics identifies monopolin: a kinetochore protein required for segregation of homologs during meiosis I.Cell. 2000; 103: 1155-1168Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). If so, which kinase phosphorylates Rec8? In mitotic yeast cells, cohesin cleavage is promoted through the phosphorylation of Scc1 by polo-like kinase (PLK, Cdc5 in yeast) (Alexandru et al., 2001Alexandru G. Uhlmann F. Mechtler K. Poupart M.A. Nasmyth K. Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast.Cell. 2001; 105: 459-472Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, Hornig and Uhlmann, 2004Hornig N.C. Uhlmann F. Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase.EMBO J. 2004; 23: 3144-3153Crossref PubMed Scopus (76) Google Scholar), which also participates in the phosphorylation of Rec8 (Clyne et al., 2003Clyne R.K. Katis V.L. Jessop L. Benjamin K.R. Herskowitz I. Lichten M. Nasmyth K. Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I.Nat. Cell Biol. 2003; 5: 480-485Crossref PubMed Scopus (176) Google Scholar, Lee and Amon, 2003Lee B.H. Amon A. Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation.Science. 2003; 300: 482-486Crossref PubMed Scopus (196) Google Scholar). Surprisingly, replacement by alanine of Rec8 residues thought to be phophorylated by Cdc5 has little or no effect on the kinetics of cohesin cleavage at meiosis I (Brar et al., 2006Brar G.A. Kiburz B.M. Zhang Y. Kim J.E. White F. Amon A. Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis.Nature. 2006; 441: 532-536Crossref PubMed Scopus (108) Google Scholar). Either Cdc5 is not the kinase responsible for promoting Rec8 cleavage or separase might after all be PP2A's real target. To address these key issues, which are fundamental to our understanding of meiosis, we have analyzed Rec8 phosphorylation without making any assumption about the kinase responsible. We show that substitution of 24 phosphorylated residues by alanine greatly hinders cleavage, whereas substitution of a subset of these with aspartate, mimicking the effects of phosphorylation, causes precocious loss of sister centromere cohesion. In addition, we show that casein kinase 1δ/ɛ (CK1δ/ɛ, Hrr25 in yeast) and Dbf4-dependent Cdc7 kinase (DDK), and not Cdc5, are essential for Rec8 cleavage. Our data suggest that shugoshins protect centromeric cohesin by opposing Rec8's phosphorylation by CK1δ/ɛ and DDK. A tandem affinity purification (TAP) tag was used to isolate Rec8 from extracts of diploid yeast cells arrested in metaphase of meiosis I. Purified proteins were analyzed by gel electrophoresis (see Figure S1A available online) or digested in solution with different proteases for mass spectrometric peptide identification. In addition to Rec8, we detected the cohesin subunits Smc1, Smc3, Scc3, and Pds5 (Table S1). Consistent with previous work (Matos et al., 2008Matos J. Lipp J.J. Bogdanova A. Guillot S. Okaz E. Junqueira M. Shevchenko A. Zachariae W. Dbf4-dependent Cdc7 kinase links DNA replication to the segregation of homologous chromosomes in meiosis I.Cell. 2008; 135: 662-678Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, Petronczki et al., 2006Petronczki M. Matos J. Mori S. Gregan J. Bogdanova A. Schwickart M. Mechtler K. Shirahige K. Zachariae W. Nasmyth K. Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1.Cell. 2006; 126: 1049-1064Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), Rec8 was associated with the protein kinases Cdc5/PLK and Hrr25/CK1δ/ɛ. Interestingly, Rec8 also copurified with the meiosis-specific recombination proteins Dmc1 and Hop1. Analysis of Rec8 peptides covering 95% of the sequence revealed phosphorylation of eight serine or threonine residues. Two Ser-Ser sequences carried a phosphate group on either one of the two residues (Figure 1A , blue residues; Figure S1A). If Rec8 phosphorylation were important for its cleavage by separase, mutation of these to alanine, which cannot be phosphorylated, should block the meiosis I division. However, substitution of all 12 residues has little effect on the meiotic progression of homozygous rec8-12A cells (data not shown). There are two possible explanations for this finding: either the phosphorylation of Rec8 is unimportant, or additional residues are phosphorylated when primary sites are mutated. To investigate the latter explanation, we mapped phosphorylation sites within Rec8-12A purified from meiotic cells. This revealed nine phosphorylated residues and one phosphate group in each of three Ser-Ser or Thr-Thr sequences (Figure 1A, green residues; Figure S1B). These additional residues were also mutated to alanine, and the resulting Rec8-24A protein was subjected to a third round of phosphosite mapping, which uncovered two more phosphorylated residues (Figure 1A, orange residues; Figure S1C). Interestingly, all 26 phosphorylation sites are located within the central region of Rec8, which is poorly, if at all, conserved among kleisins. Of these phosphorylation sites, nine were not identified in a previous analysis of Rec8 (Brar et al., 2006Brar G.A. Kiburz B.M. Zhang Y. Kim J.E. White F. Amon A. Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis.Nature. 2006; 441: 532-536Crossref PubMed Scopus (108) Google Scholar). To analyze the consequences of preventing Rec8's phosphorylation, we used live imaging to observe GFP-tagged versions of Rec8 or Rec8-24A together with Pds1-RFP and the spindle pole body (SPB) component Cnm67-RFP (Figure 1B). In addition, we measured protein levels by immunoblot analysis of protein extracts (Figure S2A). Most of the Rec8-GFP disappears from chromatin at the same time as Pds1-RFP destruction and SPB segregation, after which Rec8-GFP persists exclusively as faint “centromeric” clusters associated with each spindle pole until disappearing from view as centromeres disperse around the time spindle poles separate during meiosis II. Strikingly, Rec8-24A-GFP persists throughout the nucleus long after Pds1 destruction and remains at high levels even after SPB reduplication and separation in meiosis II (Figure 1B). Homozygous rec8-24A-GFP cells separate SPBs, express proteins required to enter metaphase I, and degrade Pds1-RFP with kinetics comparable to that of REC8-GFP cells (Figure 1C; Figure S2A). The Rec8-24A protein does not, therefore, cause a significant delay in entry into and progression through meiosis I. Rec8-24A remains at high levels beyond meiosis I also in cells lacking Sgo1, an inhibitor of cohesin removal from chromatin (Figure S2B). We conclude that the nonphosphorylatable Rec8-24A protein resists removal from chromatin and degradation at the metaphase I-to-anaphase I transition. To investigate the role of Rec8 phosphorylation in meiotic chromosome segregation, we imaged homozygous REC8-ha and rec8-24A-ha strains containing Pds1-RFP and a tet repressor-GFP fusion (TetR-GFP), which binds to tet operators integrated at LYS2 on the arms of both chromosome II homologs (Figure 1D). After S phase, TetR-GFP bound to tetO marks all four LYS2 sister sequences, and the free fraction labels the nucleus. Recombination causes the marked LYS2 loci to coalesce into a single GFP dot during prophase I. In wild-type cells, the degradation of Pds1-RFP triggers loss of sister chromatid cohesion on chromosome arms: the LYS2-GFP dot splits into two pairs of GFP foci, which segregate into the two daughter nuclei resulting from the meiosis I division. Neither the splitting of the LYS2-GFP dot nor nuclear division occurs upon Pds1-RFP destruction in rec8-24A cells. To address whether rec8-24A hinders the resolution of chiasmata, we eliminated Spo11, the endonuclease that initiates recombination. Crucially, deletion of SPO11 restores the meiosis I, but not the meiosis II, division in rec8-24A cells (Figure 1E). This also implies that sister centromeres are properly mono-orientated at meiosis I in these cells. We conclude that separase activation fails to trigger the conversion of bivalent chromosomes to dyads in rec8-24A/rec8-24A diploids. If Rec8-24A blocked the meiosis I division due to its persistence on chromatin, it should prevent nuclear division in a dominant manner. To test this, we imaged Pds1-RFP and homozygous GFP-marked LYS2 loci in REC8-ha/REC8 or rec8-24A-ha/REC8 heterozygotes (Figure 2A ). Despite the frequent splitting of sister LYS2 sequences upon Pds1-RFP destruction, the first meiotic division fails to take place in most (73%) rec8-24A-ha/REC8 cells. Importantly, the deletion of SPO11 restores this division (Figure S2C), from which we conclude that Rec8-24A is a dominant inhibitor of chiasmata resolution. Surprisingly, nuclear division in meiosis II occurs with only a small delay, suggesting that a critical amount of sister chromatid cohesion may be required to resist spindle forces effectively. To investigate whether phosphorylation is necessary for Rec8's cleavage by separase, we used the C-terminal Myc and Ha tags to compare the abundance of Rec8 cleavage products in heterozygous REC8-myc/REC8-ha and REC8-myc/rec8-24A-ha cells (Figure 2B). To facilitate detection of the short-lived cleavage products, we synchronized our meiotic cultures by using a pachytene arrest/release protocol. After transfer to sporulation medium, cells arrest in pachytene due to a deletion of the NDT80 gene. Cells are then released to synchronously progress through meiosis I by expressing NDT80 from an estradiol-inducible promoter. In REC8-myc/REC8-ha cells, the full-length proteins of both Rec8 versions start to decline, and their cleavage products accumulate (transiently) 45 min after spindle formation (Figure 2B, left). Cleavage of wild-type Rec8-myc proceeds with similar kinetics in REC8-myc/rec8-24A-ha cells, but this is neither accompanied by a major decline in full-length Rec8-24A-ha protein nor by the appearance of Ha-tagged cleavage products, and 70% of the cells fail to undergo the first nuclear division (Figure 2B, right). Although Rec8-24A is not cleaved by separase, it does not hinder the activation of the protease. Next, we measured the association of Ha- and Myc-tagged proteins with chromatin from anaphase I cells (Figure 2C). Wild-type Rec8-ha and Rec8-myc colocalize and accumulate exclusively within pericentric chromatin surrounding each SPB. In contrast, in cells coexpressing Rec8-24A-ha and Rec8-myc, Ha-tagged protein colocalizes with the bulk of chromatin, and only Myc-tagged protein surrounds the SPBs. These data imply that Rec8-24A is neither cleaved nor removed from chromatin upon activation of separase in meiosis I. As a consequence, it inhibits meiosis I nuclear division in a dominant manner. Finally, to demonstrate that nonphosphorylatable Rec8 is a poor substrate for separase in vitro, we incubated chromatin isolated from meiotic REC8-myc/REC8-ha and REC8-myc/rec8-24A-ha cells with extracts from mitotic cells that overproduce separase (Figure 2D). Separation of these reactions into an insoluble chromatin fraction and supernatant revealed that both Rec8-myc and Rec8-ha are cleaved by wild-type separase, but not by a “catalytic-dead” version. In the presence of active separase, full-length Rec8-myc and Rec8-ha disappear from the chromatin fraction while a cleavage product appears in the supernatant. Rec8-24A, in contrast, is poorly cleaved and remains in the chromatin pellet, even when wild-type Rec8-myc is readily cleaved by separase in the same extract. These data suggest that the cleavability of Rec8 depends on its phosphorylation status rather than on any meiosis-specific regulation of separase. The finding that Rec8 phosphorylation promotes its cleavage suggests that cohesin's persistence at centromeres until meiosis II might be conferred by Rec8's selective dephosphorylation by PP2A at this location. If so, replacement of serines or threonines whose phosphorylation promotes cleavage by a phosphomimetic residue such as aspartate might confer phosphorylation-independent cleavage. PP2A should not protect the phosphomimetic form, which would be cleaved at centromeres at the same time as cleavage along chromosome arms, leading to precocious sister centromere separation and nondisjunction at meiosis II. To test this, we replaced with aspartate the 12 serines and threonines from our first round of phosphosite mapping plus 2 residues close to the separase cleavage sites, creating the rec8-14D allele. In addition, we created rec8-D mutants with different subsets of these substitutions (Figure 3A ). To analyze sister chromatid cohesion, one copy of chromosome V was marked with GFP at the URA3 locus, 35 kb from the centromere. Due to monopolin activity, sister centromeres segregate to the same pole at anaphase I in 90% of wild-type cells, a phenomenon unaltered by any of the rec8-D mutations (Figure 3B). In contrast, rec8-14D and, to a lesser extent, rec8-7D-I and rec8-4D cause a noticeable increase in the frequency of anaphase I cells with separated sister URA3 sequences (Figure 3B). rec8-14D, rec8-7D-I, and rec8-4D also cause large increases in the frequency of sister centromere nondisjunction at anaphase II (40%, 33%, and 24%, respectively; Figure 3C). We conclude that phosphomimetic substitutions within Rec8's N-terminal half cause the precocious separation of sister centromeres. To test whether this phenotype is due to the cleavage of centromeric cohesin at meiosis I, we used immunofluorescence microscopy to detect Rec8 in metaphase II cells. Rec8 is observed in the vicinity of SPBs in most wild-type cells (98%), but only rarely in rec8-D mutants with phosphomimicking substitutions in the N terminus (rec8-14D, 2%; rec8-7D-I, 14%; rec8-4D, 28%; Figure 3D). We also analyzed the rec8-14D allele by using live imaging. Homozygous rec8-14D-GFP cells progress through meiosis with normal kinetics, as judged by the separation of RFP-marked SPBs and the degradation of Pds1-RFP (Figure S3). However, the Rec8-14D-GFP protein fails to persist at centromeres after the degradation of Pds1 in meiosis I (Figure 3E). Importantly, the disappearance of Rec8-14D-GFP at anaphase I is abolished by the esp1-2 mutation, which inactivates separase at 34°C (Figure 3F). Rec8-14D is not, therefore, removed from chromosomes by a separase-independent mechanism (Yu and Koshland, 2005Yu H.G. Koshland D. Chromosome morphogenesis: condensin-dependent cohesin removal during meiosis.Cell. 2005; 123: 397-407Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). We conclude that phosphomimetic substitutions cause Rec8 to be cleaved by separase at centromeres as well as along chromosome arms during meiosis I. To address whether Rec8-14D is cleaved at centromeres at the same time as along chromosome arms at meiosis I, we tested whether the aspartate substitutions suppress the inability of mam1Δ cells, which lack monopolin, to undergo the first meiotic division. Sister kinetochores are pulled to opposite poles at meiosis I in mam1Δ cells but cannot disjoin due to the resistance of centromeric cohesin to separase activity. This results in an accumulation of Pds1-negative, mononucleate cells with a single bipolar spindle (Figure 4A ). A failure to protect centromeric cohesin from separase, as occurs in sgo1Δ or rts1Δ mutants, or in cells that produce Scc1 in meiosis instead of Rec8, enables mam1Δ cells to divide their nuclei at meiosis I. rec8-14D has a similar effect (Figure 4A). Due to chiasmata, sister centromeres segregate to opposite poles in only 76% of cases in rec8-14D mam1Δ cells (Figure 4B). However, the elimination of recombination by deleting SPO11 enables almost all rec8-14D mam1Δ spo11Δ cells to disjoin sister centromeres at meiosis I (Figure 4B). A corollary is that Rec8-14D is not simply defective in conferring sister centromere cohesion because if it were, efficient biorientation of sister centromeres would not be possible in rec8-14D mam1Δ spo11Δ triple mutants. Rec8-14D creates cohesion at centromeres, but it cannot persist after meiosis I separase activation. Substitution of serines and threonines by aspartate causes precocious cleavage of centromeric Rec8 either because it mimics the effect of phosphorylation, which is both necessary and sufficient to confer cleavability by separase, or because it somehow prevents the association of Sgo1 or PP2A with centromeres. If the latter were the case, PP2A's crucial substrate could be a protein other than Rec8. However, live imaging of Sgo1-GFP and the kinetochore protein Mtw1-RFP reveal similar levels of Sgo1 during metaphase I in REC8 and rec8-14D cells (Figure 4C). Likewise, the rec8-14D allele has no detectable effect on the localization of Rts1-GFP at kinetochores. The levels of Sgo1 and Rts1 at kinetochores drop markedly as cells enter anaphase I, only to increase again at metaphase II. On chromosome spreads, however, both proteins can still be detected in the vicinity of SPBs during anaphase I (Figure 4D). We conclude that the precocious cleavage of centromeric Rec8-14D at meiosis I cannot be caused by the loss of Sgo1 or PP2A from centromeres. Instead, it must be due to PP2A's inability to prevent cleavage of Rec8-14D. What kinases are responsible for the Rec8 phosphorylation necessary for its cleavage? In mitotic cells, Cdc5/PLK promotes cohesin cleavage by phosphorylating Rec8's mitotic counterpart Scc1 (Alexandru et al., 2001Alexandru G. Uhlmann F. Mechtler K. Poupart M.A. Nasmyth K. Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast.Cell. 2001; 105: 459-472Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, Hornig and Uhlmann, 2004Hornig N.C. Uhlmann F. Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase.EMBO J. 2004; 23: 3144-3153Crossref PubMed Scopus (76) Google Scholar). Because Cdc5 also regulates Rec8 phosphorylation, it has been assumed, but never demonstrated, that Cdc5 also promotes cohesin cleavage in meiosis (Brar et al., 2006Brar G.A. Kiburz B.M. Zhang Y. Kim J.E. White F. Amon A. Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis.Nature. 2006; 441: 532-536Crossref PubMed Scopus (108) Google Scholar, Clyne et al., 2003Clyne R.K. Katis V.L. Jessop L. Benjamin K.R. Herskowitz I. Lichten M. Nasmyth K. Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I.Nat. Cell Biol. 2003; 5: 480-485Crossref PubMed Scopus (176) Google Scholar, Lee and Amon, 2003Lee B.H. Amon A. Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation.Science. 2003; 300: 482-486Crossref PubMed Scopus (196) Google Scholar). However, certain mutations allow for the efficient cleavage of Rec8 prior to Cdc5's appearance. For example, mnd2Δ ndt80Δ cells activate the meiosis-specific APC/C-Ama1 prematurely due to the absence of the APC/C inhibitor Mnd2. This causes separase activation and Rec8 cleavage in the absence of Cdc5, whose accumulation depends on Ndt80 (Oelschlaegel et al., 2005Oelschlaegel T. Schwickart M. Matos J. Bogdanova A. Camasses A. Havlis J. Shevchenko A. Zachariae W. The yeast APC/C subunit Mnd2 prevents premature sister chromatid separation triggered by the meiosis-specific APC/C-Ama1.Cell. 2005; 120: 773-788Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Penkner et al., 2005Penkner A.M. Prinz S. Ferscha S. Klein F. Mnd2, an essential antagonist of the anaphase-promoting complex during meiotic prophase.Cell. 2005; 120: 789-801Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Phosphorylation of Rec8 by Cdc5 is not, therefore, obligatory for cleavage, and other protein kinases must be involve