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Activity of the APCCdh1 form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p

Activity of the APCCdh1 form of the anaphase-promoting complex persists until S phase and... JCB Article Cdh1 Activity of the APC form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p 1,2,3 1,2 1,2 1,2 1,2 James N. Huang, Iha Park, Eric Ellingson, Laurie E. Littlepage, and David Pellman 1 2 Department of Pediatric Oncology, The Dana-Farber Cancer Institute, and Department of Pediatric Hematology, The Children’s Hospital, Harvard Medical School, Boston, MA 02115 Texas Children’s Cancer Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 ell cycle progression is driven by waves of cyclin ex- inactivated in a graded manner and is not extinguished until Cdh1 pression coupled with regulated protein degradation. S phase. Complete inactivation of APC requires S phase Cdh1 C An essential step for initiating mitosis is the inactiva- cyclins. Further, persistent APC activity throughout G1 tion of proteolysis mediated by the anaphase-promoting helps to ensure the proper timing of Cdc20p expression. complex/cyclosome (APC/C) bound to its regulator Cdh1p/ This suggests that S phase cyclins have an important role in Cdh1 Hct1p. Yeast APC was proposed previously to be inacti- allowing the accumulation of mitotic cyclins and further Cdh1 vated at Start by G1 cyclin/cyclin-dependent kinase (CDK). suggests a regulatory loop among S phase cyclins, APC , Cdh1 Cdc20 Here, we demonstrate that in a normal cell cycle APC is and APC . Introduction The anaphase-promoting complex/cyclosome (APC/C)* is a nents that are proposed to be substrate-specific APC/C acti- conserved multi-component ubiquitin ligase, which controls vators (Schwab et al., 1997; Visintin et al., 1997). Based on the proteolysis of several key cell cycle proteins (Irniger et its association with either of these two activators, the APC/C Cdc20 al., 1995; King et al., 1995; Sudakin et al., 1995; Tugend- is thought to have two functionally distinct forms: APC Cdh1 reich et al., 1995; for reviews see Peters, 1999; Zachariae and and APC (Peters, 1998). Nasmyth, 1999). In budding yeast, these include at least To coordinate a normal cell cycle, these two APC/C activ- four of the six yeast B-type cyclins (Clb1p–Clb3p and ities must be linked either directly or indirectly to cyclin- Cdh1 Clb5p) (Irniger et al., 1995; Irniger and Nasmyth, 1997; dependent kinase (CDK) activity. For APC , the linkage Schwab et al., 1997), Dbf4, a regulator of DNA replication is thought to take place primarily through CDK phosphoryla- (Cheng et al., 1999; Oshiro et al., 1999; Ferreira et al., tion of Cdh1p. In yeast, Cdh1p levels are constant through- 2000), the anaphase inhibitor Pds1p (Cohen-Fix et al., out the cell cycle, but its binding to APC/C is blocked by 1996), the polo-like kinase Cdc5p (Charles et al., 1998), the CDK phosphorylation (Zachariae et al., 1998; Jaspersen et Cdh1 APC/C regulator Cdc20p (Prinz et al., 1998; Shirayama et al., 1999). At the end of mitosis, APC is activated be- al., 1998), the checkpoint kinase Hsl (Burton and Solomon, cause Cdh1p is dephosphorylated and binds APC/C. Both 2000), and the spindle midzone protein Ase1p (Juang et al., the drop in mitotic kinase activity and the activation of the 1997). The tryptophan aspartic acid repeat proteins Cdc20p phosphatase Cdc14p contribute to Cdh1p dephosphoryla- and Cdh1p/Hct1p are substoichiometric APC/C compo- tion during mitotic exit (Visintin et al., 1998; Shou et al., Cdh1 1999). Eventually, APC is inactivated in the next cell cy- Address correspondence to David Pellman, Dana-Farber Cancer Insti- cle by rising CDK activity. However, the precise timing of tute/Harvard Medical School, Dept. of Pediatric Oncology, Rm. Cdh1 APC inactivation and the CDK isoforms involved have M612A, 44 Binney St., Boston, MA 02115. Tel.: (617) 632-4918. Fax: not been determined clearly. The experimental evidence to (617) 632-5757. E-mail: [email protected] date supports the view that G1 CDKs alone inactivate what *Abbreviations used in this paper: APC/C, anaphase-promoting complex/ Cdh1 is now known to be APC (Amon et al., 1994). Addition- cyclosome; CDK, cyclin-dependent kinase; db, destruction box; GST, glu- ally, the possibility that S phase cyclins could have a role in tathione S-transferase; HA, hemagglutinin; YEP, yeast extract and peptone. Cdh1 APC inactivation has been raised because failure to de- Key words: cell cycle; cyclin-dependent kinases; mitosis; S phase; ubiquitin grade an S phase cyclin by the end of mitosis can block  The Rockefeller University Press, 0021-9525/2001/07/85/10 $5.00 The Journal of Cell Biology, Volume 154, Number 1, July 9, 2001 85–94 http://www.jcb.org/cgi/doi/10.1083/jcb.200102007 85 86 The Journal of Cell Biology | Volume 154, 2001 Cdh1 APC function and mitotic exit (Shirayama et al., 1999; cdc4 mutant, which arrests before S phase with high G1 Zachariae and Nasmyth, 1999). CDK activity and high levels of Sic1p. We found that Ase1p Cdh1 During G1, APC activity is required to prevent ex- is rapidly degraded at the cdc4 block with similar kinetics to pression of proteins that may interfere with bud emergence, that observed previously in -factor–arrested cells (Fig. 1 A; lead to premature DNA replication, or disturb spindle as- Juang et al., 1997). The 5–10 min half-life of Ase1p at the sembly (Amon et al., 1994; Irniger and Nasmyth, 1997; cdc4 arrest point contrasts sharply with the 60 min half- Juang et al., 1997). Before the onset of the ensuing mitosis, life of Ase1p observed in cycling or nocodazole-arrested cells Cdh1 APC must be turned off in order to allow its mitotic (Juang et al., 1997; data not shown). Ase1p is also rapidly substrates to accumulate. Several lines of evidence linked G1 degraded in a skp1-11 strain, which also arrests in late G1 CDK activity (Cln1p-Cln3p bound to Cdk1p) to the inacti- (data not shown). This also contrasts with the degradation Cdh1 Cdh1 vation of APC . First, in cdc4-arrested cells, which block of the APC substrate Clb2p, which is stable at the cdc4 in late G1 with high levels of G1 CDK activity, ectopically block (Amon et al., 1994; Amon, 1997; unpublished data; expressed M phase B-type cyclin Clb2p was stable, and its see Fig. 4 B, glu). Like Ase1p degradation in -factor– stability required the expression of G1 cyclins (Amon et al., arrested cells, Ase1p degradation at the cdc4 block required 1994; Amon, 1997). Second, the accumulation of a consti- Cdh1p and the destruction box (db), a cis-acting sequence tutively expressed Clb2p correlated with the rise in G1 cy- for APC/C-mediated degradation (Fig. 1, A and B). There- Cdh1 clin expression that occurs in G1 (Amon et al., 1994). Fi- fore, Ase1p is rapidly degraded by APC in the presence nally, Cdh1p was heavily phosphorylated and presumed to of high levels of G1 CDK activity. be inactive in late G1-arrested cells (Zachariae et al., 1998). The rapid degradation of Ase1p in cdc4-arrested cells Together, these experiments suggested that G1 CDK phos- prompted us to determine if Cdh1p could bind APC/C in phorylation of Cdh1p was the mechanism for inactivating cdc4-arrested cells. Unphosphorylated Cdh1p binds to APC/ Cdh1 APC . However, these experiments were potentially com- C strongly, whereas CDK-phosphorylated Cdh1p does not plicated by the fact that Clb2p is not only an APC/C sub- (Zachariae et al., 1998; Jaspersen et al., 1999). Cdh1p was strate but also an APC/C regulator whose expression might found previously to be heavily phosphorylated in cells ar- influence its own stability (Amon, 1997). rested in late G1 (Zachariae et al., 1998). We also found Cdh1 Unexpectedly, we found that another APC substrate, that Cdh1p appears to be phosphorylated in cdc4-arrested the microtubule-binding protein Ase1p, was rapidly de- cells, although judging from its mobility it appears to be less graded in late G1 (cdc4 block). Further, Ase1p degradation phosphorylated than in nocodazole-arrested cells (Fig. 1 C, Cdh1 Cdh1 at this arrest point had the hallmarks of APC -dependent input). Consistent with the apparent activity of APC at proteolysis: it required both the Ase1p destruction box and the cdc4 block, Cdh1p was coimmunoprecipitated with Cdh1 Cdh1p. A minimal Ase1p sequence for APC -mediated APC/C in cdc4-arrested cells. The coimmunoprecipitation Cdh1 degradation was defined whose constitutive expression did experiments suggest that the amount of APC diminishes not affect cell cycle progression. Using this degradation sig- in a graded manner during G1: high levels of Cdh1p coim- Cdh1 nal to monitor APC activity in vivo, we found that munoprecipitate with Cdc16p in -factor–arrested cells, low Cdh1 APC is inactivated during S phase and that the S phase levels of Cdh1p coimmunoprecipitate in cdc4-arrested cells, Cdh1 cyclin Clb5p was required for the normal timing of APC and coimmunoprecipitation is undetectable in nocodazole- inactivation. Further, we show that the degradation of arrested cells (Fig. 1 C). Cdh1 Cdc20p in late G1 is also APC dependent and that pre- Many of the known APC/C substrates are also APC/C mature expression of Cdc20p delays the progression through regulators (Amon, 1997; Charles et al., 1998; Shirayama et S phase. These findings suggest that both G1 and S phase al., 1998). This complicates the in vivo analysis of the pro- Cdh1 Cdh1 cyclins are required to shut off APC and that APC teolysis of these proteins because of the possibility that ec- activity in G1 plays an important role in ensuring the proper topic expression of these proteins during half-life measure- timing of Cdc20p expression. ments could itself alter APC/C function (Amon, 1997). Ase1p is a microtubule-binding and cross-linking protein that is required for the structural integrity of the anaphase Results spindle (Juang et al., 1997; unpublished data). Although In budding yeast, genetic and biochemical analyses have de- Ase1p is not expected to regulate APC/C, high levels of fined several molecular events necessary for transit through Ase1p activate the spindle assembly checkpoint and thereby G1 and into S phase. CDK activity is low in early G1 but might indirectly affect APC/C activity (Juang et al., 1997; rises in late G1 when G1 cyclins are expressed. The rise in for review see Amon, 1999). To create an inert reporter of Cdh1 G1 CDK activity induces apical bud growth and initiates a APC activity, we constructed chimeras between Ase1p cascade of events that leads to the phosphorylation and sub- and glutathione S-transferase (GST). These chimeras en- sequent degradation of the B-type cyclin/CDK inhibitor abled us to define the minimal Ase1p sequence necessary for Cdh1 Sic1p. Sic1p degradation permits the activation of S phase APC -mediated degradation. The half-lives of the GST– CDKs (Cdk1p bound to Clb5p and Clb6p, two of the six Ase1p fusions were measured in -factor–arrested cells (Fig. B-type cyclins in yeast) and therefore the initiation of S 2 A). A chimera containing the COOH-terminal 254 amino phase (for review see Krek, 1998). acids (R632-I885) of Ase1p was degraded with similar ki- To determine if Ase1p destruction was inactivated by G1 netics to full-length Ase1p. Further deletions up to amino CDK activity, the half-life of Ase1p was determined in a acid 802 from the COOH-terminal end of R632-I885 were Cdh1 S phase inactivation of APC | Huang et al. 87 prise the minimal sequence for Ase1p degradation in -fac- tor–arrested cells. The degradation of the GST–R632-I885 fusion protein (hereafter referred to as C254) was characterized in more de- tail and found to have the characteristics of a bona fide Cdh1 APC substrate. Like wild-type Ase1p, C254 degradation in -factor–arrested cells required Cdc23p and hence APC/ C function (Fig. 2 B). C254 degradation in -factor– arrested cells also required the Ase1p destruction box and Cdh1p but was not affected by loss of Cdc20p (data not shown). Also, like wild-type Ase1p this fusion was stable in both S and G2/M phase–arrested cells (data not shown). Fi- nally, as shown above for wild-type Ase1p, C254 was rapidly degraded in cdc4-arrested cells, and this degradation re- quired the db and Cdh1p (Fig. 2, C and D). Thus, the deg- radation of C254 closely mimics that of Ase1p. The localization of full-length Ase1p is restricted to the spindle midzone (the zone of overlap between the two half- spindles) (Pellman et al., 1995). By contrast, the C254 fu- sion protein localized diffusely throughout the nucleus and as expected did not complement an ase1 mutation (Fig. 2 E; data not shown). Therefore, the specific localization of Ase1p on the mitotic spindle is not required for the normal timing of its degradation. Cdh1 The finding that Ase1p was degraded by APC at the cdc4 block suggested that G1 CDK activity was not suffi- Cdh1 Cdh1 cient to fully inactivate APC . To monitor APC activ- ity during a normal cell cycle, C254 was expressed from the GALL promoter (Mumberg et al., 1994), a derivative of the GAL1,10 promoter that is transcribed at low levels through- out the cell cycle when cells are grown in galactose-contain- ing medium. Importantly, constitutive expression of C254 did not affect cell growth (data not shown) or cell cycle pro- gression (Fig. 3 A). Because transcription of C254 from this promoter was constant during the cell cycle (Fig. 3 B), the steady-state level of C254 protein reflected its degradation. Analysis of C254 levels in synchronized cells suggested Cdh1 Cdh1 Figure 1. APC is active in late G1. (A) cdc4 strains containing that APC is inactivated during S phase. Wild-type cells GAL1,10::ASE1 or GAL1,10::ASE1-DB (Juang et al., 1997) were ar- expressing C254 were released from a G1 block, and C254 rested in yeast extract and peptone (YEP) raffinose at 36C. Expres- levels were determined at intervals after release. Cell cycle sion from the GAL1,10 promoter was induced for 60 min, and the position was monitored by Sic1p degradation, which occurs half-life of Ase1p or Ase1p-db was determined. G1 arrest was con- firmed by FACS . (B) The half-life of Ase1p was determined in ar- at the G1/S transition (Schwob et al., 1994), by the level of rested cdc4 cdh1 clb6 and cdc4 CDH1 clb6 strains as above. histone 2B mRNA, which is induced at S phase onset (Here- The clb6 mutation was introduced into these strains to promote ef- ford et al., 1981), and by FACS analysis. We found that ficient arrest. cdc4 cdh1 double mutants arrest poorly in late G1, C254 accumulated after Sic1p was degraded, when histone probably because of persistent low levels of B-type cyclins from the 2B mRNA peaked and when FACS analysis demonstrated previous mitosis (unpublished data). (C) cdc4 strains containing an that cells had entered S phase (Fig. 3 B). epitope-tagged APC/C subunit (Cdc16p-myc6), or the untagged Cdh1 control, and HA-tagged Cdh1p (HA3-Cdh1p) were arrested at 36C, Because APC appeared to be inactivated during S and APC/C was immunoprecipitated using a monoclonal antibody phase, we determined if removal of Clb5p, the major S directed against the myc epitope. Extracts and immunoprecipitates phase cyclin (Epstein and Cross, 1992; Schwob and Na- were probed with 12CA5 to detect HA3-Cdh1p. smyth, 1993), would affect the accumulation of C254. Con- Cdh1 sistent with a role for Clb5p in APC inactivation, C254 accumulation was delayed by 10 min in a clb5 strain also rapidly degraded in -factor–arrested cells. By contrast, (Fig. 3 C). By contrast, deletion of CLB3 and CLB4, which deletion of 22 amino acids on the NH -terminal side of the encode M phase cyclins, had no effect on the timing of R632-I885 peptide resulted in a stable chimera (T654-I885; C254 destruction (data not shown). Therefore, the normal Cdh1 Fig. 2 A). 7 of these 22 residues are lysines, raising the possi- timing of APC inactivation requires Clb5p. bility that these may be the site(s) of Ase1p ubiquitination. We considered the possibility that ectopic expression of Together, these results suggest that residues 632–802 com- Clb2p could overcome the high levels of Sic1p in late G1- 88 The Journal of Cell Biology | Volume 154, 2001 Cdh1 Figure 2. Ase1p domain sufficient for APC -mediated degrada- tion. (A) Stability of Ase1p NH -terminal truncations fused to GST in -factor–arrested cells. Chimeras were expressed from the GALL promoter for 30 min in -factor–arrested cells, and the half-lives of the chimeras were assayed. R632-I885 is the COOH-terminal 254 amino acids of Ase1p. (B) CDC23 and cdc23-1 strains containing GAL1,10::C254 were arrested with -factor, shifted to 36C for 30 min to inactivate Cdc23p, then galactose was added for 30 min to induce expression of C254, and the half-life of the chimera was de- termined. (C) cdc4 cells containing C254 or a db mutant of C254 (C254-db) were arrested at 36C, and the half-lives of the fusion pro- teins were determined 30 min after the addition of galactose. (D) The half-life of C254 was determined in arrested cdc4 cdh1 clb6 and cdc4 CDH1 clb6 cells. (E) Cells constitutively expressing C254 (the GST–R632-I885 fusion protein) were prepared for immunofluo- rescence. No signal was detected in this strain grown under repress- ing conditions (glucose; data not shown). C254 is diffusely localized to the nucleus and appears to be at higher levels in mitotic cells. Cdh1 S phase inactivation of APC | Huang et al. 89 Cdh1 Figure 3. APC activity is inactivated during S phase. (A) Con- stitutive expression of C254 does not alter the cell cycle. Strains constitutively expressing either GST or GST–R632-I885 (C254) from the GALL promoter by growth in galactose-containing medium were arrested with -factor, collected by filtration, and released into the cell cycle. FACS analysis was performed at the indicated time points after release. (B) S phase stabilization of C254. A wild-type strain expressing C254 from the GALL promoter was grown in galactose-containing medium and arrested in -factor and released into fresh medium. Samples were then collected for Western blotting, Northern blotting, and FACS analysis at the indicated time points. (C) CLB5 and clb5 strains expressing C254 under the control of the GALL promoter were grown in galactose-containing medium, arrested in -factor, re- leased, and samples collected as in B. arrested cells and combine with G1 CDK activity to inacti- of Clb2p in cdc4-arrested cells in the presence or absence of Cdh1 vate APC . This could explain the difference between the Sic1p overexpression. In the absence of Sic1p overexpres- timing of degradation of Ase1p and Clb2p. To test this idea, sion, Clb2p has a half-life of 60 min, whereas with coover- cdc4 cells constitutively expressing low levels of Clb2p were expression of Sic1p, Clb2p half-life is 20 min. FACS synchronized with -factor and released at 36C to the cdc4 analysis showed that the cells remained arrested in G1 for arrest point in the presence or absence of hemagglutinin 30 min after the samples were collected (Fig. 4 B). To- (HA)-tagged Sic1p overexpressed from the GAL1,10 pro- gether, our results suggest that a combination of G1 cyclins moter (Verma et al., 1997). As expected, Clb2p accumu- and either S phase or M phase cyclins are required to inacti- Cdh1 lated in the control culture (Fig. 4 A, glu). However, Clb2p vate APC . Cdh1 accumulation was prevented in the Sic1p-overexpressing The persistence of APC activity through late G1 sug- Cdh1 culture (Fig. 4, compare F with gal). Northern blot analy- gested that the degradation of certain APC substrates sis showed that CLB2 mRNA levels did not differ between might be required for the normal execution of S phase. One the two cultures (Fig. 4 A). We next measured the half-life appealing candidate for such a substrate is the APC activator 90 The Journal of Cell Biology | Volume 154, 2001 Figure 4. Overexpression of Sic1p in- creases Clb2p turnover at the cdc4 block. (A) Overexpression of Sic1 blocks Clb2p accumulation at the cdc4 block. A cdc4 strain expressing Clb2p-HA3 from the methionine-repressible MET25 (Mumberg et al., 1994) promoter and Sic1p-HA from the GAL1,10 promoter (Verma et al., 1997) were arrested with -factor and released at the nonpermis- sive temperature to arrest at the cdc4 block. After release, the culture was split, and Sic1p-HA expression was in- duced by the addition of galactose (gal) in one culture and repressed in the other with glucose (glu). The two cultures were kept at 36C, until the cells were arrested with elongated buds and G1 DNA content. The levels of Clb2p-HA3 and Sic1p-HA were determined by Western blotting, and CLB2 mRNA lev- els were determined by Northern blot- ting. The MET25 promoter expresses at low levels even under the repressing conditions of this experiment (medium containing methionine). (B) Sic1p over- expression restores Clb2p degradation in cdc4-arrested cells. The strain used in A was treated as above, and the half-life of Clb2p-HA3 was determined after ex- pression was shut off by the addition of methionine and cycloheximide. Cdh1 Cdc20p. Metazoan Cdc20p is an APC substrate (Pfleger expressing strain was delayed in transiting S phase (Fig. 5 B). and Kirschner, 2000; Sorensen et al., 2000), and premature Next, we tested whether this delay in transiting S phase is Cdc20 activation of APC might interfere with the expression of S due to the failure to degrade Cdc20p in G1 by comparing phase cyclins. Indeed, we found that degradation of yeast the cell cycle transit of strains expressing either wild-type Cdh1 Cdc20p is APC dependent in cdc4-arrested cells (Fig. 5 A). Cdc20p or Cdc20Box12. Compared with the Cdc20p- To test the functional consequences of premature accumu- expressing strain, the Cdc20Box12 strain was also de- lation of Cdc20p during G1, we determined if inappropriate layed in transiting S phase (Fig. 5 C). This delay correlates expression of Cdc20p in G1 affected cell cycle progression with the amount of Cdc20p that is expressed in G1 because through S phase. Cells expressing Cdc20Box12-HA Cdc20p was expressed at lower levels than Cdc20Box12 from the pGAL1,10 promoter were released into galactose- until cells reached S phase (Fig. 5 C). These experiments sug- Cdh1 containing medium from a G1 block, and cell cycle transit gest that APC activity during G1 helps to ensure the was monitored by FACS analysis. By contrast with the con- proper timing of Cdc20p expression and thereby enables S trol strain containing vector only, the Cdc20Box12- phase progression to proceed with normal kinetics. Cdh1 S phase inactivation of APC | Huang et al. 91 Cdh1 man Cdh1p, APC promotes the degradation of human Discussion Cdh1 Cdc20p (Pfleger and Kirschner, 2000; Sorensen et al., We found that APC is inactivated during S phase, and 2000). Our finding that yeast Cdc20p is also degraded by its complete inactivation requires Clb5p. Both Ase1p and Cdh1 APC in late G1 demonstrates that this regulatory mecha- Cdc20p were degraded in late G1-arrested cells containing nism is conserved. Further, our in vivo experiments suggest high levels of G1 CDK activity. Cdh1p was required for the that inappropriate expression of Cdc20p can delay progres- degradation of both substrates. We also found that a fraction sion through S phase. Therefore, we propose that the follow- of Cdh1p was bound to the APC/C in late G1-arrested cells. Cdh1 ing regulation occurs at the G1/S transition. APC is ac- Further, the S phase cyclin Clb5p was required for the nor- tive throughout G1. Clb5p accumulates in late G1, but Cdh1 mal timing of APC inactivation. Thus, in a normal cell Clb5/CDK activity is held in check by the presence of Sic1p cycle the additive activities of G1 and S phase CDKs inacti- (Schwob et al., 1994). After Sic1p is degraded, Clb5/CDK Cdh1 vate APC . These findings have two implications for the Cdh1 activity drives DNA replication and inactivates APC . Be- design of the yeast cell cycle. First, the key role for Clb5p in Cdh1 Cdh1 cause Cdc20p is an APC substrate, APC activity en- Cdh1 APC inactivation suggests that Clb5p has an important Cdc20 sures that no Cdc20p is present to activate APC until role in enabling the expression of mitotic cyclins. This func- after Clb5/CDK is active. It should be emphasized that tion was previously ascribed entirely to G1 cyclins. Second, Cdh1 Cdc20 degradation of Cdc20p by APC is one of several controls because Clb5p is degraded by APC our finding that on Cdc20p expression as CDC20 transcription is also low in Cdh1 yeast Cdc20p is an APC substrate suggests that high Cdh1 G1 (Prinz et al., 1998; Shirayama et al., 1998). Additionally, APC activity throughout G1 may help ensure that Cdc20 full APC activation also appears to require the phos- Clb5p can accumulate sufficiently to drive a normal S phase. phorylation of the APC/C itself (Kotani et al., 1999; Shtein- Our conclusions differ from that of previous work using berg et al., 1999; Kramer et al., 2000; Rudner and Murray, Cdh1 Clb2p degradation to monitor APC activity in vivo. The 2000). The existence of multiple overlapping control mecha- prior work suggested that G1 CDKs alone could inactivate nisms is a common theme in the regulation of cell cycle tran- Cdh1 what is now known to be APC (Amon et al., 1994). The sitions. We make the analogy to the regulation of mitotic exit difference in the timing of Clb2p, Ase1p and Cdc20p degra- where proteolysis, decreased transcription, and the expression dation reported here could either be due to intrinsic differ- Cdh1 of an inhibitor all collaborate to shut off mitotic cyclins. ences in how these proteins are recognized by APC or to Finally, our findings impact on the physiological role effects from the ectopic expression of Clb2p. Although we Cdc20 of Clb5p degradation by APC . Mutant cells lacking found that some Cdh1p is bound to APC/C in cdc4-arrested Cdc20p but able to separate sister chromatids (cdc20 cells, considerably less is bound than in -factor–arrested Cdh1 pds1 strains) cannot exit from mitosis (Shirayama et al., cells. It is therefore possible that APC is a more efficient 1999). However, these cells are able to exit mitosis if CLB5 enzyme for Ase1p and Cdc20p ubiquitination than for is deleted. Based on these findings, it was suggested that Clb2p ubiquitination. However, we also found that coover- Cdc20p promotes mitotic exit in a normal cell cycle by de- expression of Sic1p at the cdc4 arrest point blocks Clb2p ac- grading Clb5p, which in turn releases inhibition of Sic1p cumulation and restores Clb2p degradation in cdc4-arrested and Cdh1p. The results presented here raise the possibility cells. This suggests that ectopic Clb/CDK activity was re- Cdh1 that the role of Clb5p degradation in mitotic exit is indirect sponsible for turning off the APC in the previous experi- and occurs by suppression of mitotic cyclins. In the absence ments. We therefore favor the idea that both G1- and Cdh1 of Clb5p, APC may remain partially active, resulting in B-type cyclin–associated kinases are required to fully inacti- decreased levels of the mitotic cyclins Clb1p and Clb2p. In Cdh1 vate APC . Clb2p expressed in late G1 could either di- Cdh1 Cdh1 cells with reduced levels of mitotic cyclins, APC alone rectly inactivate the APC or compete for Sic1p binding may be sufficient to trigger mitotic exit. The latter model is Cdh1 to S phase cyclins thereby freeing them to inhibit APC . consistent with the established role of mitotic cyclin proteol- How might G1 and S phase CDKs cooperate to inactivate ysis in mitotic exit. Cdh1 APC ? Despite the presence of highly phosphorylated Cdh1 The APC/C is strikingly conserved among eukaryotes. Cdh1p at the cdc4 block, enough APC remains for rapid The conservation of the APC/C is seen not only in the pep- degradation of Ase1p and Cdc20p. Since Cdh1p has multi- tide sequence of its components and substrates but also ex- ple functionally important CDK phosphorylation sites (Za- tends to aspects of its regulation. Our results highlight what chariae et al., 1998; Jaspersen et al., 1999), G1 and S phase may be a general role for S phase cyclins in regulating CDK might preferentially phosphorylate different sites or Cdh1 APC . In human cells, the S phase cyclin A complexed to make an additive contribution to the total level of Cdh1p Cdk1p phosphorylates and inactivates Cdh1p during S phosphorylation. Alternatively, the inhibitory effects of S phase (Lukas et al., 1999). Thus, S phase inactivation of phase CDK activity could be through phosphorylation of Cdh1 APC appears to be a conserved mechanism for control of another substrate such as subunits of APC (Rudner and the cell cycle. Murray, 2000). Whatever the mechanism for Clb5p-medi- Cdh1 ated inactivation of APC , these results identify a novel Cdh1 Clb5p role in the inactivation of APC and by inference Materials and methods in the accumulation of mitotic cyclins. Strains and microbial techniques These experiments also have implications for the rela- Media and genetic techniques were as described (Sherman et al., 1986). To Cdh1 Cdc20 tionship among Clb5p, APC , and APC . In both arrest cells in late G1, cdc4-1 cells were shifted to 36C until 90% of cells showed either multiple or elongated buds. To arrest cells in S or G2/M the Xenopus extract system and in cells overexpressing hu- 92 The Journal of Cell Biology | Volume 154, 2001 Cdh1 Figure 5. APC regulation of Cdc20p expression. (A) Cdh1 Cdc20p is an APC substrate in late G1. Expression of HA- tagged Cdc20p from the GAL1,10 promoter (Prinz et al., 1998) was induced for 40 min, and the half-life of Cdc20p was determined in arrested cdc4 cdh1 clb6 and cdc4 CDH1 clb6 strains as for Ase1p in the legend to Fig. 1 B. (B) Expression of stable Cdc20p in G1 delays S phase progres- sion. Wild-type strains containing either vector alone or GAL1,10::Cdc20Box12-HA (Prinz et al., 1998) were ar- rested with -factor in YEP raffinose 3%. Expression from the GAL1,10 promoter was induced for 30 min, and then cells were washed and released into YEP galactose 3%. (C) Inabil- ity to degrade Cdc20p in G1 delays S phase progression. Wild-type strains containing either GAL1,10::Cdc20-HA or GAL1,10::Cdc20Box12-HA (Prinz et al., 1998) were ar- rested with -factor in YEP raffinose 3%. Expression from the GAL1,10 promoter was induced for 15 min, and then cells were washed and released into YEP galactose 2%. The levels of the HA-tagged Cdc20p and Cdc20Box12 were deter- mined by Western blotting and quantified as described in Materials and methods. phase, hydroxyurea or nocodazole was added to a final concentration of from the MET25 promoter, methionine (final concentration 1 mM) and cy- 10 mg/ml or 15 g/ml, respectively. To arrest cells in early G1, -factor cloheximide were added. Protein samples were prepared from cells col- was added to a final concentration of 100 nM for sst1 strains and 5 M for lected at the indicated time points and analyzed by Western blotting. SST1 strains. To release cells from -factor arrest, cells were collected by Protein extracts were made as described (Yaffe and Schatz, 1984). filtration, washed, and resuspended in fresh medium. All strains were de- Immunoblots were developed using ECL. Ase1p and derivatives were de- rivatives of W303 except the A364-derived clb5 (a gift from F. Cross, The tected with either 9E10 (Evan et al., 1985) for myc-tagged proteins or a Rockefeller University, New York, NY) and CLB5 control strains in Fig. 3 C. rabbit polyclonal anti-Ase1p antibody (Juang et al., 1997). myc-tagged Cdc16p was detected with 9E10. HA-tagged Clb2p, Cdh1p, and Cdc20p Mutagenesis and cloning were detected with the 12CA5 monoclonal antibody (Field et al., 1988). DNA manipulations were performed as described (Sambrook et al., 1989). Polyclonal antibodies were used to detect -tubulin (Serotec), -tubulin (a Ase1p truncations were generated by PCRs. The sequence of the oligonu- gift from F. Solomon, Massachusetts Institute of Technology, Cambridge, cleotide primers used for PCR are available upon request. All PCR-gener- MA), and Sic1p (a gift from J.W. Harper, Baylor College of Medicine, ated constructs were verified by DNA sequencing. Houston, TX). For quantification in Fig. 4, immunoblots were developed with ECL, fluorescence was detected using StormImager (Molecular Dy- Half-life determination namics), and signal intensities were measure using ImageQuant 5.0 soft- Cells were arrested in medium containing 3% raffinose; galactose was ware. For quantification in Fig. 5, immunoblots were probed with an added to a final concentration of 3% for 30–60 min, and then glucose (fi- IRDye800 fluorescent-labeled secondary antibody and quantitated on the nal concentration 3%) and cycloheximide (final concentration 1 mg/ml) Odyssey Infrared Imaging System (Li-Cor); fluorescence between blots was were added to shut off transcription and translation. For proteins expressed normalized to reference standards that were loaded on both blots. Cdh1 S phase inactivation of APC | Huang et al. 93 Figure 5 (continued) Submitted: 1 February 2001 Immunoprecipitation Revised: 11 May 2001 Strains containing myc-tagged Cdc16p (or untagged control) and HA- Accepted: 25 May 2001 tagged Cdh1p were arrested at either 36C (-factor, cdc4) or 24C (no- codazole). Cells were lysed using glass beads in IP buffer (50 mM Hepes, pH 7.3, 150 mM potassium acetate, 10 mM sodium fluoride, 1 mM EDTA, 1 mM PMSF, 20 mM -glycerophosphate, 1 mM sodium vanadate, Phos- References phatase Inhibitor sets I and II [Calbiochem], 0.1% Triton X-100, and Com- plete™ protease inhibitors [Roche]). Extracts (2–4 mg) were incubated with Amon, A. 1997. Regulation of B-type cyclin proteolysis by Cdc28-associated ki- 9E10 monoclonal antibodies (1,000:1) in IP buffer for 2 h at 4C. Anti- nases in budding yeast. EMBO J. 16:2693–2702. body-bound extracts were then incubated with protein G–plus agarose Amon, A. 1999. The spindle checkpoint. Curr. Opin. Genet. Dev. 9:69–75. beads (Calbiochem) for 4 h at 4C in IP buffer. The beads were washed five Amon, A., S. Irniger, and K. Nasmyth. 1994. Closing the cell cycle circle in yeast: times with IP buffer and resuspended in sample buffer. Bound proteins G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 were analyzed by SDS-PAGE and immunoblotting. cyclins in the next cycle. Cell. 77:1037–1050. Burton, J.L., and M.J. Solomon. 2000. Hsl1p, a Swe1p inhibitor, is degraded via Flow cytometry the anaphase-promoting complex. Mol. Cell. Biol. 20:4614–4625. Cells were prepared for FACS analysis as described (Pellman et al., 1995) Charles, J.F., S.L. Jaspersen, R.L. Tinker-Kulberg, L. Hwang, A. Szidon, and D.O. and analyzed on a FACScan™ using CellQuest software (Becton Dickin- Morgan. 1998. The Polo-related kinase Cdc5 activates and is destroyed by son). the mitotic cyclin destruction machinery in S. cerevisiae. Curr. Biol. 8:497– Northern blotting Cheng, L., T. Collyer, and C.F. Hardy. 1999. Cell cycle regulation of DNA repli- RNA preparation, Northern blot analysis, and the probes to detect histone cation initiator factor Dbf4p. Mol. Cell. 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Diffley. 2000. Dbf4p, an es- We thank A. Amon, F. Cross, R. Deshaies, W. Harper, S. Jentsch, D. Mor- sential S phase-promoting factor, is targeted for degradation by the ana- gan, K. Nasmyth, F. Solomon, and W. Zachariae for strains and/or re- phase-promoting complex. Mol. Cell. Biol. 20:242–248. agents; Yue-Li Juang for making the original observation that Ase1p is de- Field, J., J. Nikawa, D. Broek, B. MacDonald, L. Rodgers, I.A. Wilson, R.A. graded in cdc4-arrested cells; A. Amon and members of the Pellman lab Lerner, and M. Wigler. 1988. Purification of a RAS-responsive adenylyl cy- for discussions; A. Amon, M. Christman, P. de Figueiredo, M. McLaughlin, clase complex from Saccharomyces cerevisiae by use of an epitope addition S. Milligan, D. Pati, and S. Plon for comments on the manuscript, and par- method. Mol. Cell. Biol. 8:2159–2165. ticular thanks to S. Plon for her support. Hereford, L.M., M.A. Osley, T.R. Ludwig, and C.S. McLaughlin. 1981. 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Genes Dev. 7:1160– Jaspersen, S.L., J.F. Charles, and D.O. Morgan. 1999. Inhibitory phosphorylation 1175. of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phos- Schwob, E., T. Bohm, M.D. Mendenhall, and K. Nasmyth. 1994. The B-type cy- phatase Cdc14. Curr. Biol. 9:227–236. clin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Juang, Y.L., J. Huang, J.M. Peters, M.E. McLaughlin, C.Y. Tai, and D. Pellman. Cell. 79:233–244. 1997. APC-mediated proteolysis of Ase1 and the morphogenesis of the mi- Sherman, F., J.B. Hicks, and G.R. Fink. 1986. Methods in Yeast Genetics. Cold totic spindle. Science. 275:1311–1314. Spring Harbor Laboratory, Cold Spring Harbor, New York. 198 pp. King, R.W., J.M. Peters, S. Tugendreich, M. Rolfe, P. Hieter, and M.W. Kirsch- Shirayama, M., W. Zachariae, R. Ciosk, and K. Nasmyth. 1998. The Polo-like ki- ner. 1995. A 20S complex containing CDC27 and CDC16 catalyzes the mi- nase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and tosis-specific conjugation of ubiquitin to cyclin B. Cell. 81:279–288. substrates of the anaphase promoting complex in Saccharomyces cerevisiae. Kotani, S., H. Tanaka, H. Yasuda, and K. Todokoro. 1999. Regulation of APC ac- EMBO J. 17:1336–1349. (Cdc20) tivity by phosphorylation and regulatory factors. J. Cell Biol. 146:791–800. Shirayama, M., A. Toth, M. Galova, and K. Nasmyth. 1999. APC promotes Kramer, E.R., N. Scheuringer, A.V. Podtelejnikov, M. Mann, and J.M. Peters. exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5. 2000. Mitotic regulation of the APC activator proteins CDC20 and CDH1. Nature. 402:203–207. Mol. Biol. Cell. 11:1555–1569. Shou, W., J.H. Seol, A. Shevchenko, C. Baskerville, D. Moazed, Z.W. Chen, J. Krek, W. 1998. Proteolysis and the G1-S transition: the SCF connection. Curr. Jang, H. Charbonneau, and R.J. Deshaies. 1999. Exit from mitosis is trig- Opin. Genet. Dev. 8:36–42. gered by Tem1-dependent release of the protein phosphatase Cdc14 from Lukas, C., C.S. Sorensen, E. Kramer, E. Santoni-Rugiu, C. Lindeneg, J.M. Peters, nucleolar RENT complex. Cell. 97:233–244. J. Bartek, and J. Lukas. 1999. Accumulation of cyclin B1 requires E2F and Shteinberg, M., Y. Protopopov, T. Listovsky, M. Brandeis, and A. Hershko. 1999. cyclin-A-dependent rearrangement of the anaphase-promoting complex. Phosphorylation of the cyclosome is required for its stimulation by Fizzy/ Nature. 401:815–818. cdc20. Biochem. Biophys. Res. Commun. 260:193–198. Mumberg, D., R. Muller, and M. Funk. 1994. Regulatable promoters of Saccharo- Sorensen, C.S., C. Lukas, E.R. Kramer, J.M. Peters, J. Bartek, and J. Lukas. 2000. myces cerevisiae: comparison of transcriptional activity and their use for het- Nonperiodic activity of the human anaphase-promoting complex-cdh1 erologous expression. Nucleic Acids Res. 22:5767–5768. ubiquitin ligase results in continuous DNA synthesis uncoupled from mito- Oshiro, G., J.C. Owens, Y. Shellman, R.A. Sclafani, and J.J. Li. 1999. Cell cycle sis. Mol. Cell. Biol. 20:7613–7623. control of Cdc7p kinase activity through regulation of Dbf4p stability. Mol. Sudakin, V., D. Ganoth, A. Dahan, H. Heller, J. Hershko, F.C. Luca, J.V. Ruder- Cell. Biol. 19:4888–4896. man, and A. Hershko. 1995. The cyclosome, a large complex containing cy- Pellman, D., M. Bagget, Y.H. Tu, G.R. Fink, and H. Tu. 1995. Two microtubule- clin-selective ubiquitin ligase activity, targets cyclins for destruction at the associated proteins required for anaphase spindle movement in Saccharomy- end of mitosis. Mol. Biol. Cell. 6:185–197. ces cerevisiae. J. Cell Biol. 130:1373–1385. Tugendreich, S., J. Tomkiel, W. Earnshaw, and P. Hieter. 1995. CDC27Hs colo- Peters, J.M. 1998. 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Activity of the APCCdh1 form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p

Huang, James N.; Park, Iha; Ellingson, Eric; Littlepage, Laurie E.; Pellman, David
The Journal of Cell Biology , Volume 154 (1) – Jul 9, 2001
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Copyright © 2001, The Rockefeller University Press
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10.1083/jcb.200102007
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JCB Article Cdh1 Activity of the APC form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p 1,2,3 1,2 1,2 1,2 1,2 James N. Huang, Iha Park, Eric Ellingson, Laurie E. Littlepage, and David Pellman 1 2 Department of Pediatric Oncology, The Dana-Farber Cancer Institute, and Department of Pediatric Hematology, The Children’s Hospital, Harvard Medical School, Boston, MA 02115 Texas Children’s Cancer Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 ell cycle progression is driven by waves of cyclin ex- inactivated in a graded manner and is not extinguished until Cdh1 pression coupled with regulated protein degradation. S phase. Complete inactivation of APC requires S phase Cdh1 C An essential step for initiating mitosis is the inactiva- cyclins. Further, persistent APC activity throughout G1 tion of proteolysis mediated by the anaphase-promoting helps to ensure the proper timing of Cdc20p expression. complex/cyclosome (APC/C) bound to its regulator Cdh1p/ This suggests that S phase cyclins have an important role in Cdh1 Hct1p. Yeast APC was proposed previously to be inacti- allowing the accumulation of mitotic cyclins and further Cdh1 vated at Start by G1 cyclin/cyclin-dependent kinase (CDK). suggests a regulatory loop among S phase cyclins, APC , Cdh1 Cdc20 Here, we demonstrate that in a normal cell cycle APC is and APC . Introduction The anaphase-promoting complex/cyclosome (APC/C)* is a nents that are proposed to be substrate-specific APC/C acti- conserved multi-component ubiquitin ligase, which controls vators (Schwab et al., 1997; Visintin et al., 1997). Based on the proteolysis of several key cell cycle proteins (Irniger et its association with either of these two activators, the APC/C Cdc20 al., 1995; King et al., 1995; Sudakin et al., 1995; Tugend- is thought to have two functionally distinct forms: APC Cdh1 reich et al., 1995; for reviews see Peters, 1999; Zachariae and and APC (Peters, 1998). Nasmyth, 1999). In budding yeast, these include at least To coordinate a normal cell cycle, these two APC/C activ- four of the six yeast B-type cyclins (Clb1p–Clb3p and ities must be linked either directly or indirectly to cyclin- Cdh1 Clb5p) (Irniger et al., 1995; Irniger and Nasmyth, 1997; dependent kinase (CDK) activity. For APC , the linkage Schwab et al., 1997), Dbf4, a regulator of DNA replication is thought to take place primarily through CDK phosphoryla- (Cheng et al., 1999; Oshiro et al., 1999; Ferreira et al., tion of Cdh1p. In yeast, Cdh1p levels are constant through- 2000), the anaphase inhibitor Pds1p (Cohen-Fix et al., out the cell cycle, but its binding to APC/C is blocked by 1996), the polo-like kinase Cdc5p (Charles et al., 1998), the CDK phosphorylation (Zachariae et al., 1998; Jaspersen et Cdh1 APC/C regulator Cdc20p (Prinz et al., 1998; Shirayama et al., 1999). At the end of mitosis, APC is activated be- al., 1998), the checkpoint kinase Hsl (Burton and Solomon, cause Cdh1p is dephosphorylated and binds APC/C. Both 2000), and the spindle midzone protein Ase1p (Juang et al., the drop in mitotic kinase activity and the activation of the 1997). The tryptophan aspartic acid repeat proteins Cdc20p phosphatase Cdc14p contribute to Cdh1p dephosphoryla- and Cdh1p/Hct1p are substoichiometric APC/C compo- tion during mitotic exit (Visintin et al., 1998; Shou et al., Cdh1 1999). Eventually, APC is inactivated in the next cell cy- Address correspondence to David Pellman, Dana-Farber Cancer Insti- cle by rising CDK activity. However, the precise timing of tute/Harvard Medical School, Dept. of Pediatric Oncology, Rm. Cdh1 APC inactivation and the CDK isoforms involved have M612A, 44 Binney St., Boston, MA 02115. Tel.: (617) 632-4918. Fax: not been determined clearly. The experimental evidence to (617) 632-5757. E-mail: [email protected] date supports the view that G1 CDKs alone inactivate what *Abbreviations used in this paper: APC/C, anaphase-promoting complex/ Cdh1 is now known to be APC (Amon et al., 1994). Addition- cyclosome; CDK, cyclin-dependent kinase; db, destruction box; GST, glu- ally, the possibility that S phase cyclins could have a role in tathione S-transferase; HA, hemagglutinin; YEP, yeast extract and peptone. Cdh1 APC inactivation has been raised because failure to de- Key words: cell cycle; cyclin-dependent kinases; mitosis; S phase; ubiquitin grade an S phase cyclin by the end of mitosis can block  The Rockefeller University Press, 0021-9525/2001/07/85/10 $5.00 The Journal of Cell Biology, Volume 154, Number 1, July 9, 2001 85–94 http://www.jcb.org/cgi/doi/10.1083/jcb.200102007 85 86 The Journal of Cell Biology | Volume 154, 2001 Cdh1 APC function and mitotic exit (Shirayama et al., 1999; cdc4 mutant, which arrests before S phase with high G1 Zachariae and Nasmyth, 1999). CDK activity and high levels of Sic1p. We found that Ase1p Cdh1 During G1, APC activity is required to prevent ex- is rapidly degraded at the cdc4 block with similar kinetics to pression of proteins that may interfere with bud emergence, that observed previously in -factor–arrested cells (Fig. 1 A; lead to premature DNA replication, or disturb spindle as- Juang et al., 1997). The 5–10 min half-life of Ase1p at the sembly (Amon et al., 1994; Irniger and Nasmyth, 1997; cdc4 arrest point contrasts sharply with the 60 min half- Juang et al., 1997). Before the onset of the ensuing mitosis, life of Ase1p observed in cycling or nocodazole-arrested cells Cdh1 APC must be turned off in order to allow its mitotic (Juang et al., 1997; data not shown). Ase1p is also rapidly substrates to accumulate. Several lines of evidence linked G1 degraded in a skp1-11 strain, which also arrests in late G1 CDK activity (Cln1p-Cln3p bound to Cdk1p) to the inacti- (data not shown). This also contrasts with the degradation Cdh1 Cdh1 vation of APC . First, in cdc4-arrested cells, which block of the APC substrate Clb2p, which is stable at the cdc4 in late G1 with high levels of G1 CDK activity, ectopically block (Amon et al., 1994; Amon, 1997; unpublished data; expressed M phase B-type cyclin Clb2p was stable, and its see Fig. 4 B, glu). Like Ase1p degradation in -factor– stability required the expression of G1 cyclins (Amon et al., arrested cells, Ase1p degradation at the cdc4 block required 1994; Amon, 1997). Second, the accumulation of a consti- Cdh1p and the destruction box (db), a cis-acting sequence tutively expressed Clb2p correlated with the rise in G1 cy- for APC/C-mediated degradation (Fig. 1, A and B). There- Cdh1 clin expression that occurs in G1 (Amon et al., 1994). Fi- fore, Ase1p is rapidly degraded by APC in the presence nally, Cdh1p was heavily phosphorylated and presumed to of high levels of G1 CDK activity. be inactive in late G1-arrested cells (Zachariae et al., 1998). The rapid degradation of Ase1p in cdc4-arrested cells Together, these experiments suggested that G1 CDK phos- prompted us to determine if Cdh1p could bind APC/C in phorylation of Cdh1p was the mechanism for inactivating cdc4-arrested cells. Unphosphorylated Cdh1p binds to APC/ Cdh1 APC . However, these experiments were potentially com- C strongly, whereas CDK-phosphorylated Cdh1p does not plicated by the fact that Clb2p is not only an APC/C sub- (Zachariae et al., 1998; Jaspersen et al., 1999). Cdh1p was strate but also an APC/C regulator whose expression might found previously to be heavily phosphorylated in cells ar- influence its own stability (Amon, 1997). rested in late G1 (Zachariae et al., 1998). We also found Cdh1 Unexpectedly, we found that another APC substrate, that Cdh1p appears to be phosphorylated in cdc4-arrested the microtubule-binding protein Ase1p, was rapidly de- cells, although judging from its mobility it appears to be less graded in late G1 (cdc4 block). Further, Ase1p degradation phosphorylated than in nocodazole-arrested cells (Fig. 1 C, Cdh1 Cdh1 at this arrest point had the hallmarks of APC -dependent input). Consistent with the apparent activity of APC at proteolysis: it required both the Ase1p destruction box and the cdc4 block, Cdh1p was coimmunoprecipitated with Cdh1 Cdh1p. A minimal Ase1p sequence for APC -mediated APC/C in cdc4-arrested cells. The coimmunoprecipitation Cdh1 degradation was defined whose constitutive expression did experiments suggest that the amount of APC diminishes not affect cell cycle progression. Using this degradation sig- in a graded manner during G1: high levels of Cdh1p coim- Cdh1 nal to monitor APC activity in vivo, we found that munoprecipitate with Cdc16p in -factor–arrested cells, low Cdh1 APC is inactivated during S phase and that the S phase levels of Cdh1p coimmunoprecipitate in cdc4-arrested cells, Cdh1 cyclin Clb5p was required for the normal timing of APC and coimmunoprecipitation is undetectable in nocodazole- inactivation. Further, we show that the degradation of arrested cells (Fig. 1 C). Cdh1 Cdc20p in late G1 is also APC dependent and that pre- Many of the known APC/C substrates are also APC/C mature expression of Cdc20p delays the progression through regulators (Amon, 1997; Charles et al., 1998; Shirayama et S phase. These findings suggest that both G1 and S phase al., 1998). This complicates the in vivo analysis of the pro- Cdh1 Cdh1 cyclins are required to shut off APC and that APC teolysis of these proteins because of the possibility that ec- activity in G1 plays an important role in ensuring the proper topic expression of these proteins during half-life measure- timing of Cdc20p expression. ments could itself alter APC/C function (Amon, 1997). Ase1p is a microtubule-binding and cross-linking protein that is required for the structural integrity of the anaphase Results spindle (Juang et al., 1997; unpublished data). Although In budding yeast, genetic and biochemical analyses have de- Ase1p is not expected to regulate APC/C, high levels of fined several molecular events necessary for transit through Ase1p activate the spindle assembly checkpoint and thereby G1 and into S phase. CDK activity is low in early G1 but might indirectly affect APC/C activity (Juang et al., 1997; rises in late G1 when G1 cyclins are expressed. The rise in for review see Amon, 1999). To create an inert reporter of Cdh1 G1 CDK activity induces apical bud growth and initiates a APC activity, we constructed chimeras between Ase1p cascade of events that leads to the phosphorylation and sub- and glutathione S-transferase (GST). These chimeras en- sequent degradation of the B-type cyclin/CDK inhibitor abled us to define the minimal Ase1p sequence necessary for Cdh1 Sic1p. Sic1p degradation permits the activation of S phase APC -mediated degradation. The half-lives of the GST– CDKs (Cdk1p bound to Clb5p and Clb6p, two of the six Ase1p fusions were measured in -factor–arrested cells (Fig. B-type cyclins in yeast) and therefore the initiation of S 2 A). A chimera containing the COOH-terminal 254 amino phase (for review see Krek, 1998). acids (R632-I885) of Ase1p was degraded with similar ki- To determine if Ase1p destruction was inactivated by G1 netics to full-length Ase1p. Further deletions up to amino CDK activity, the half-life of Ase1p was determined in a acid 802 from the COOH-terminal end of R632-I885 were Cdh1 S phase inactivation of APC | Huang et al. 87 prise the minimal sequence for Ase1p degradation in -fac- tor–arrested cells. The degradation of the GST–R632-I885 fusion protein (hereafter referred to as C254) was characterized in more de- tail and found to have the characteristics of a bona fide Cdh1 APC substrate. Like wild-type Ase1p, C254 degradation in -factor–arrested cells required Cdc23p and hence APC/ C function (Fig. 2 B). C254 degradation in -factor– arrested cells also required the Ase1p destruction box and Cdh1p but was not affected by loss of Cdc20p (data not shown). Also, like wild-type Ase1p this fusion was stable in both S and G2/M phase–arrested cells (data not shown). Fi- nally, as shown above for wild-type Ase1p, C254 was rapidly degraded in cdc4-arrested cells, and this degradation re- quired the db and Cdh1p (Fig. 2, C and D). Thus, the deg- radation of C254 closely mimics that of Ase1p. The localization of full-length Ase1p is restricted to the spindle midzone (the zone of overlap between the two half- spindles) (Pellman et al., 1995). By contrast, the C254 fu- sion protein localized diffusely throughout the nucleus and as expected did not complement an ase1 mutation (Fig. 2 E; data not shown). Therefore, the specific localization of Ase1p on the mitotic spindle is not required for the normal timing of its degradation. Cdh1 The finding that Ase1p was degraded by APC at the cdc4 block suggested that G1 CDK activity was not suffi- Cdh1 Cdh1 cient to fully inactivate APC . To monitor APC activ- ity during a normal cell cycle, C254 was expressed from the GALL promoter (Mumberg et al., 1994), a derivative of the GAL1,10 promoter that is transcribed at low levels through- out the cell cycle when cells are grown in galactose-contain- ing medium. Importantly, constitutive expression of C254 did not affect cell growth (data not shown) or cell cycle pro- gression (Fig. 3 A). Because transcription of C254 from this promoter was constant during the cell cycle (Fig. 3 B), the steady-state level of C254 protein reflected its degradation. Analysis of C254 levels in synchronized cells suggested Cdh1 Cdh1 Figure 1. APC is active in late G1. (A) cdc4 strains containing that APC is inactivated during S phase. Wild-type cells GAL1,10::ASE1 or GAL1,10::ASE1-DB (Juang et al., 1997) were ar- expressing C254 were released from a G1 block, and C254 rested in yeast extract and peptone (YEP) raffinose at 36C. Expres- levels were determined at intervals after release. Cell cycle sion from the GAL1,10 promoter was induced for 60 min, and the position was monitored by Sic1p degradation, which occurs half-life of Ase1p or Ase1p-db was determined. G1 arrest was con- firmed by FACS . (B) The half-life of Ase1p was determined in ar- at the G1/S transition (Schwob et al., 1994), by the level of rested cdc4 cdh1 clb6 and cdc4 CDH1 clb6 strains as above. histone 2B mRNA, which is induced at S phase onset (Here- The clb6 mutation was introduced into these strains to promote ef- ford et al., 1981), and by FACS analysis. We found that ficient arrest. cdc4 cdh1 double mutants arrest poorly in late G1, C254 accumulated after Sic1p was degraded, when histone probably because of persistent low levels of B-type cyclins from the 2B mRNA peaked and when FACS analysis demonstrated previous mitosis (unpublished data). (C) cdc4 strains containing an that cells had entered S phase (Fig. 3 B). epitope-tagged APC/C subunit (Cdc16p-myc6), or the untagged Cdh1 control, and HA-tagged Cdh1p (HA3-Cdh1p) were arrested at 36C, Because APC appeared to be inactivated during S and APC/C was immunoprecipitated using a monoclonal antibody phase, we determined if removal of Clb5p, the major S directed against the myc epitope. Extracts and immunoprecipitates phase cyclin (Epstein and Cross, 1992; Schwob and Na- were probed with 12CA5 to detect HA3-Cdh1p. smyth, 1993), would affect the accumulation of C254. Con- Cdh1 sistent with a role for Clb5p in APC inactivation, C254 accumulation was delayed by 10 min in a clb5 strain also rapidly degraded in -factor–arrested cells. By contrast, (Fig. 3 C). By contrast, deletion of CLB3 and CLB4, which deletion of 22 amino acids on the NH -terminal side of the encode M phase cyclins, had no effect on the timing of R632-I885 peptide resulted in a stable chimera (T654-I885; C254 destruction (data not shown). Therefore, the normal Cdh1 Fig. 2 A). 7 of these 22 residues are lysines, raising the possi- timing of APC inactivation requires Clb5p. bility that these may be the site(s) of Ase1p ubiquitination. We considered the possibility that ectopic expression of Together, these results suggest that residues 632–802 com- Clb2p could overcome the high levels of Sic1p in late G1- 88 The Journal of Cell Biology | Volume 154, 2001 Cdh1 Figure 2. Ase1p domain sufficient for APC -mediated degrada- tion. (A) Stability of Ase1p NH -terminal truncations fused to GST in -factor–arrested cells. Chimeras were expressed from the GALL promoter for 30 min in -factor–arrested cells, and the half-lives of the chimeras were assayed. R632-I885 is the COOH-terminal 254 amino acids of Ase1p. (B) CDC23 and cdc23-1 strains containing GAL1,10::C254 were arrested with -factor, shifted to 36C for 30 min to inactivate Cdc23p, then galactose was added for 30 min to induce expression of C254, and the half-life of the chimera was de- termined. (C) cdc4 cells containing C254 or a db mutant of C254 (C254-db) were arrested at 36C, and the half-lives of the fusion pro- teins were determined 30 min after the addition of galactose. (D) The half-life of C254 was determined in arrested cdc4 cdh1 clb6 and cdc4 CDH1 clb6 cells. (E) Cells constitutively expressing C254 (the GST–R632-I885 fusion protein) were prepared for immunofluo- rescence. No signal was detected in this strain grown under repress- ing conditions (glucose; data not shown). C254 is diffusely localized to the nucleus and appears to be at higher levels in mitotic cells. Cdh1 S phase inactivation of APC | Huang et al. 89 Cdh1 Figure 3. APC activity is inactivated during S phase. (A) Con- stitutive expression of C254 does not alter the cell cycle. Strains constitutively expressing either GST or GST–R632-I885 (C254) from the GALL promoter by growth in galactose-containing medium were arrested with -factor, collected by filtration, and released into the cell cycle. FACS analysis was performed at the indicated time points after release. (B) S phase stabilization of C254. A wild-type strain expressing C254 from the GALL promoter was grown in galactose-containing medium and arrested in -factor and released into fresh medium. Samples were then collected for Western blotting, Northern blotting, and FACS analysis at the indicated time points. (C) CLB5 and clb5 strains expressing C254 under the control of the GALL promoter were grown in galactose-containing medium, arrested in -factor, re- leased, and samples collected as in B. arrested cells and combine with G1 CDK activity to inacti- of Clb2p in cdc4-arrested cells in the presence or absence of Cdh1 vate APC . This could explain the difference between the Sic1p overexpression. In the absence of Sic1p overexpres- timing of degradation of Ase1p and Clb2p. To test this idea, sion, Clb2p has a half-life of 60 min, whereas with coover- cdc4 cells constitutively expressing low levels of Clb2p were expression of Sic1p, Clb2p half-life is 20 min. FACS synchronized with -factor and released at 36C to the cdc4 analysis showed that the cells remained arrested in G1 for arrest point in the presence or absence of hemagglutinin 30 min after the samples were collected (Fig. 4 B). To- (HA)-tagged Sic1p overexpressed from the GAL1,10 pro- gether, our results suggest that a combination of G1 cyclins moter (Verma et al., 1997). As expected, Clb2p accumu- and either S phase or M phase cyclins are required to inacti- Cdh1 lated in the control culture (Fig. 4 A, glu). However, Clb2p vate APC . Cdh1 accumulation was prevented in the Sic1p-overexpressing The persistence of APC activity through late G1 sug- Cdh1 culture (Fig. 4, compare F with gal). Northern blot analy- gested that the degradation of certain APC substrates sis showed that CLB2 mRNA levels did not differ between might be required for the normal execution of S phase. One the two cultures (Fig. 4 A). We next measured the half-life appealing candidate for such a substrate is the APC activator 90 The Journal of Cell Biology | Volume 154, 2001 Figure 4. Overexpression of Sic1p in- creases Clb2p turnover at the cdc4 block. (A) Overexpression of Sic1 blocks Clb2p accumulation at the cdc4 block. A cdc4 strain expressing Clb2p-HA3 from the methionine-repressible MET25 (Mumberg et al., 1994) promoter and Sic1p-HA from the GAL1,10 promoter (Verma et al., 1997) were arrested with -factor and released at the nonpermis- sive temperature to arrest at the cdc4 block. After release, the culture was split, and Sic1p-HA expression was in- duced by the addition of galactose (gal) in one culture and repressed in the other with glucose (glu). The two cultures were kept at 36C, until the cells were arrested with elongated buds and G1 DNA content. The levels of Clb2p-HA3 and Sic1p-HA were determined by Western blotting, and CLB2 mRNA lev- els were determined by Northern blot- ting. The MET25 promoter expresses at low levels even under the repressing conditions of this experiment (medium containing methionine). (B) Sic1p over- expression restores Clb2p degradation in cdc4-arrested cells. The strain used in A was treated as above, and the half-life of Clb2p-HA3 was determined after ex- pression was shut off by the addition of methionine and cycloheximide. Cdh1 Cdc20p. Metazoan Cdc20p is an APC substrate (Pfleger expressing strain was delayed in transiting S phase (Fig. 5 B). and Kirschner, 2000; Sorensen et al., 2000), and premature Next, we tested whether this delay in transiting S phase is Cdc20 activation of APC might interfere with the expression of S due to the failure to degrade Cdc20p in G1 by comparing phase cyclins. Indeed, we found that degradation of yeast the cell cycle transit of strains expressing either wild-type Cdh1 Cdc20p is APC dependent in cdc4-arrested cells (Fig. 5 A). Cdc20p or Cdc20Box12. Compared with the Cdc20p- To test the functional consequences of premature accumu- expressing strain, the Cdc20Box12 strain was also de- lation of Cdc20p during G1, we determined if inappropriate layed in transiting S phase (Fig. 5 C). This delay correlates expression of Cdc20p in G1 affected cell cycle progression with the amount of Cdc20p that is expressed in G1 because through S phase. Cells expressing Cdc20Box12-HA Cdc20p was expressed at lower levels than Cdc20Box12 from the pGAL1,10 promoter were released into galactose- until cells reached S phase (Fig. 5 C). These experiments sug- Cdh1 containing medium from a G1 block, and cell cycle transit gest that APC activity during G1 helps to ensure the was monitored by FACS analysis. By contrast with the con- proper timing of Cdc20p expression and thereby enables S trol strain containing vector only, the Cdc20Box12- phase progression to proceed with normal kinetics. Cdh1 S phase inactivation of APC | Huang et al. 91 Cdh1 man Cdh1p, APC promotes the degradation of human Discussion Cdh1 Cdc20p (Pfleger and Kirschner, 2000; Sorensen et al., We found that APC is inactivated during S phase, and 2000). Our finding that yeast Cdc20p is also degraded by its complete inactivation requires Clb5p. Both Ase1p and Cdh1 APC in late G1 demonstrates that this regulatory mecha- Cdc20p were degraded in late G1-arrested cells containing nism is conserved. Further, our in vivo experiments suggest high levels of G1 CDK activity. Cdh1p was required for the that inappropriate expression of Cdc20p can delay progres- degradation of both substrates. We also found that a fraction sion through S phase. Therefore, we propose that the follow- of Cdh1p was bound to the APC/C in late G1-arrested cells. Cdh1 ing regulation occurs at the G1/S transition. APC is ac- Further, the S phase cyclin Clb5p was required for the nor- tive throughout G1. Clb5p accumulates in late G1, but Cdh1 mal timing of APC inactivation. Thus, in a normal cell Clb5/CDK activity is held in check by the presence of Sic1p cycle the additive activities of G1 and S phase CDKs inacti- (Schwob et al., 1994). After Sic1p is degraded, Clb5/CDK Cdh1 vate APC . These findings have two implications for the Cdh1 activity drives DNA replication and inactivates APC . Be- design of the yeast cell cycle. First, the key role for Clb5p in Cdh1 Cdh1 cause Cdc20p is an APC substrate, APC activity en- Cdh1 APC inactivation suggests that Clb5p has an important Cdc20 sures that no Cdc20p is present to activate APC until role in enabling the expression of mitotic cyclins. This func- after Clb5/CDK is active. It should be emphasized that tion was previously ascribed entirely to G1 cyclins. Second, Cdh1 Cdc20 degradation of Cdc20p by APC is one of several controls because Clb5p is degraded by APC our finding that on Cdc20p expression as CDC20 transcription is also low in Cdh1 yeast Cdc20p is an APC substrate suggests that high Cdh1 G1 (Prinz et al., 1998; Shirayama et al., 1998). Additionally, APC activity throughout G1 may help ensure that Cdc20 full APC activation also appears to require the phos- Clb5p can accumulate sufficiently to drive a normal S phase. phorylation of the APC/C itself (Kotani et al., 1999; Shtein- Our conclusions differ from that of previous work using berg et al., 1999; Kramer et al., 2000; Rudner and Murray, Cdh1 Clb2p degradation to monitor APC activity in vivo. The 2000). The existence of multiple overlapping control mecha- prior work suggested that G1 CDKs alone could inactivate nisms is a common theme in the regulation of cell cycle tran- Cdh1 what is now known to be APC (Amon et al., 1994). The sitions. We make the analogy to the regulation of mitotic exit difference in the timing of Clb2p, Ase1p and Cdc20p degra- where proteolysis, decreased transcription, and the expression dation reported here could either be due to intrinsic differ- Cdh1 of an inhibitor all collaborate to shut off mitotic cyclins. ences in how these proteins are recognized by APC or to Finally, our findings impact on the physiological role effects from the ectopic expression of Clb2p. Although we Cdc20 of Clb5p degradation by APC . Mutant cells lacking found that some Cdh1p is bound to APC/C in cdc4-arrested Cdc20p but able to separate sister chromatids (cdc20 cells, considerably less is bound than in -factor–arrested Cdh1 pds1 strains) cannot exit from mitosis (Shirayama et al., cells. It is therefore possible that APC is a more efficient 1999). However, these cells are able to exit mitosis if CLB5 enzyme for Ase1p and Cdc20p ubiquitination than for is deleted. Based on these findings, it was suggested that Clb2p ubiquitination. However, we also found that coover- Cdc20p promotes mitotic exit in a normal cell cycle by de- expression of Sic1p at the cdc4 arrest point blocks Clb2p ac- grading Clb5p, which in turn releases inhibition of Sic1p cumulation and restores Clb2p degradation in cdc4-arrested and Cdh1p. The results presented here raise the possibility cells. This suggests that ectopic Clb/CDK activity was re- Cdh1 that the role of Clb5p degradation in mitotic exit is indirect sponsible for turning off the APC in the previous experi- and occurs by suppression of mitotic cyclins. In the absence ments. We therefore favor the idea that both G1- and Cdh1 of Clb5p, APC may remain partially active, resulting in B-type cyclin–associated kinases are required to fully inacti- decreased levels of the mitotic cyclins Clb1p and Clb2p. In Cdh1 vate APC . Clb2p expressed in late G1 could either di- Cdh1 Cdh1 cells with reduced levels of mitotic cyclins, APC alone rectly inactivate the APC or compete for Sic1p binding may be sufficient to trigger mitotic exit. The latter model is Cdh1 to S phase cyclins thereby freeing them to inhibit APC . consistent with the established role of mitotic cyclin proteol- How might G1 and S phase CDKs cooperate to inactivate ysis in mitotic exit. Cdh1 APC ? Despite the presence of highly phosphorylated Cdh1 The APC/C is strikingly conserved among eukaryotes. Cdh1p at the cdc4 block, enough APC remains for rapid The conservation of the APC/C is seen not only in the pep- degradation of Ase1p and Cdc20p. Since Cdh1p has multi- tide sequence of its components and substrates but also ex- ple functionally important CDK phosphorylation sites (Za- tends to aspects of its regulation. Our results highlight what chariae et al., 1998; Jaspersen et al., 1999), G1 and S phase may be a general role for S phase cyclins in regulating CDK might preferentially phosphorylate different sites or Cdh1 APC . In human cells, the S phase cyclin A complexed to make an additive contribution to the total level of Cdh1p Cdk1p phosphorylates and inactivates Cdh1p during S phosphorylation. Alternatively, the inhibitory effects of S phase (Lukas et al., 1999). Thus, S phase inactivation of phase CDK activity could be through phosphorylation of Cdh1 APC appears to be a conserved mechanism for control of another substrate such as subunits of APC (Rudner and the cell cycle. Murray, 2000). Whatever the mechanism for Clb5p-medi- Cdh1 ated inactivation of APC , these results identify a novel Cdh1 Clb5p role in the inactivation of APC and by inference Materials and methods in the accumulation of mitotic cyclins. Strains and microbial techniques These experiments also have implications for the rela- Media and genetic techniques were as described (Sherman et al., 1986). To Cdh1 Cdc20 tionship among Clb5p, APC , and APC . In both arrest cells in late G1, cdc4-1 cells were shifted to 36C until 90% of cells showed either multiple or elongated buds. To arrest cells in S or G2/M the Xenopus extract system and in cells overexpressing hu- 92 The Journal of Cell Biology | Volume 154, 2001 Cdh1 Figure 5. APC regulation of Cdc20p expression. (A) Cdh1 Cdc20p is an APC substrate in late G1. Expression of HA- tagged Cdc20p from the GAL1,10 promoter (Prinz et al., 1998) was induced for 40 min, and the half-life of Cdc20p was determined in arrested cdc4 cdh1 clb6 and cdc4 CDH1 clb6 strains as for Ase1p in the legend to Fig. 1 B. (B) Expression of stable Cdc20p in G1 delays S phase progres- sion. Wild-type strains containing either vector alone or GAL1,10::Cdc20Box12-HA (Prinz et al., 1998) were ar- rested with -factor in YEP raffinose 3%. Expression from the GAL1,10 promoter was induced for 30 min, and then cells were washed and released into YEP galactose 3%. (C) Inabil- ity to degrade Cdc20p in G1 delays S phase progression. Wild-type strains containing either GAL1,10::Cdc20-HA or GAL1,10::Cdc20Box12-HA (Prinz et al., 1998) were ar- rested with -factor in YEP raffinose 3%. Expression from the GAL1,10 promoter was induced for 15 min, and then cells were washed and released into YEP galactose 2%. The levels of the HA-tagged Cdc20p and Cdc20Box12 were deter- mined by Western blotting and quantified as described in Materials and methods. phase, hydroxyurea or nocodazole was added to a final concentration of from the MET25 promoter, methionine (final concentration 1 mM) and cy- 10 mg/ml or 15 g/ml, respectively. To arrest cells in early G1, -factor cloheximide were added. Protein samples were prepared from cells col- was added to a final concentration of 100 nM for sst1 strains and 5 M for lected at the indicated time points and analyzed by Western blotting. SST1 strains. To release cells from -factor arrest, cells were collected by Protein extracts were made as described (Yaffe and Schatz, 1984). filtration, washed, and resuspended in fresh medium. All strains were de- Immunoblots were developed using ECL. Ase1p and derivatives were de- rivatives of W303 except the A364-derived clb5 (a gift from F. Cross, The tected with either 9E10 (Evan et al., 1985) for myc-tagged proteins or a Rockefeller University, New York, NY) and CLB5 control strains in Fig. 3 C. rabbit polyclonal anti-Ase1p antibody (Juang et al., 1997). myc-tagged Cdc16p was detected with 9E10. HA-tagged Clb2p, Cdh1p, and Cdc20p Mutagenesis and cloning were detected with the 12CA5 monoclonal antibody (Field et al., 1988). DNA manipulations were performed as described (Sambrook et al., 1989). Polyclonal antibodies were used to detect -tubulin (Serotec), -tubulin (a Ase1p truncations were generated by PCRs. The sequence of the oligonu- gift from F. Solomon, Massachusetts Institute of Technology, Cambridge, cleotide primers used for PCR are available upon request. All PCR-gener- MA), and Sic1p (a gift from J.W. Harper, Baylor College of Medicine, ated constructs were verified by DNA sequencing. Houston, TX). For quantification in Fig. 4, immunoblots were developed with ECL, fluorescence was detected using StormImager (Molecular Dy- Half-life determination namics), and signal intensities were measure using ImageQuant 5.0 soft- Cells were arrested in medium containing 3% raffinose; galactose was ware. For quantification in Fig. 5, immunoblots were probed with an added to a final concentration of 3% for 30–60 min, and then glucose (fi- IRDye800 fluorescent-labeled secondary antibody and quantitated on the nal concentration 3%) and cycloheximide (final concentration 1 mg/ml) Odyssey Infrared Imaging System (Li-Cor); fluorescence between blots was were added to shut off transcription and translation. For proteins expressed normalized to reference standards that were loaded on both blots. Cdh1 S phase inactivation of APC | Huang et al. 93 Figure 5 (continued) Submitted: 1 February 2001 Immunoprecipitation Revised: 11 May 2001 Strains containing myc-tagged Cdc16p (or untagged control) and HA- Accepted: 25 May 2001 tagged Cdh1p were arrested at either 36C (-factor, cdc4) or 24C (no- codazole). Cells were lysed using glass beads in IP buffer (50 mM Hepes, pH 7.3, 150 mM potassium acetate, 10 mM sodium fluoride, 1 mM EDTA, 1 mM PMSF, 20 mM -glycerophosphate, 1 mM sodium vanadate, Phos- References phatase Inhibitor sets I and II [Calbiochem], 0.1% Triton X-100, and Com- plete™ protease inhibitors [Roche]). Extracts (2–4 mg) were incubated with Amon, A. 1997. Regulation of B-type cyclin proteolysis by Cdc28-associated ki- 9E10 monoclonal antibodies (1,000:1) in IP buffer for 2 h at 4C. Anti- nases in budding yeast. EMBO J. 16:2693–2702. body-bound extracts were then incubated with protein G–plus agarose Amon, A. 1999. The spindle checkpoint. Curr. Opin. Genet. Dev. 9:69–75. beads (Calbiochem) for 4 h at 4C in IP buffer. The beads were washed five Amon, A., S. Irniger, and K. Nasmyth. 1994. Closing the cell cycle circle in yeast: times with IP buffer and resuspended in sample buffer. Bound proteins G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 were analyzed by SDS-PAGE and immunoblotting. cyclins in the next cycle. Cell. 77:1037–1050. Burton, J.L., and M.J. Solomon. 2000. Hsl1p, a Swe1p inhibitor, is degraded via Flow cytometry the anaphase-promoting complex. Mol. Cell. Biol. 20:4614–4625. Cells were prepared for FACS analysis as described (Pellman et al., 1995) Charles, J.F., S.L. Jaspersen, R.L. Tinker-Kulberg, L. Hwang, A. Szidon, and D.O. and analyzed on a FACScan™ using CellQuest software (Becton Dickin- Morgan. 1998. The Polo-related kinase Cdc5 activates and is destroyed by son). the mitotic cyclin destruction machinery in S. cerevisiae. Curr. Biol. 8:497– Northern blotting Cheng, L., T. Collyer, and C.F. Hardy. 1999. Cell cycle regulation of DNA repli- RNA preparation, Northern blot analysis, and the probes to detect histone cation initiator factor Dbf4p. Mol. Cell. Biol. 19:4270–4278. 2B1 and actin mRNA were as described (Pellman et al., 1995). The probe Cohen-Fix, O., J.M. Peters, M.W. Kirschner, and D. Koshland. 1996. Anaphase for ASE1 mRNA was a 1.2-kb fragment from nucleotides 1896–3108 of the initiation in Saccharomyces cerevisiae is controlled by the APC-dependent ASE1 locus (where 1 is the A residue of the ATG codon). degradation of the anaphase inhibitor Pds1p. Genes Dev. 10:3081–3093. Epstein, C.B., and F.R. Cross. 1992. CLB5: a novel B cyclin from budding yeast Immunofluorescence with a role in S phase. Genes Dev. 6:1695–1706. Cells were fixed and prepared for DAPI staining and immunofluorescence Evan, G.I., G.K. Lewis, G. Ramsay, and J.M. Bishop. 1985. Isolation of mono- as described previously (Pringle et al., 1991). C254 containing three clonal antibodies specific for human c-myc proto-oncogene product. Mol. COOH-terminal myc tags was detected with 9E10. Cell. Biol. 5:3610–3616. Ferreira, M.F., C. Santocanale, L.S. Drury, and J.F. Diffley. 2000. Dbf4p, an es- We thank A. Amon, F. Cross, R. Deshaies, W. Harper, S. Jentsch, D. Mor- sential S phase-promoting factor, is targeted for degradation by the ana- gan, K. Nasmyth, F. Solomon, and W. Zachariae for strains and/or re- phase-promoting complex. Mol. Cell. Biol. 20:242–248. agents; Yue-Li Juang for making the original observation that Ase1p is de- Field, J., J. Nikawa, D. Broek, B. MacDonald, L. Rodgers, I.A. Wilson, R.A. graded in cdc4-arrested cells; A. Amon and members of the Pellman lab Lerner, and M. Wigler. 1988. Purification of a RAS-responsive adenylyl cy- for discussions; A. Amon, M. Christman, P. de Figueiredo, M. McLaughlin, clase complex from Saccharomyces cerevisiae by use of an epitope addition S. Milligan, D. Pati, and S. Plon for comments on the manuscript, and par- method. Mol. Cell. Biol. 8:2159–2165. ticular thanks to S. Plon for her support. Hereford, L.M., M.A. Osley, T.R. Ludwig, and C.S. McLaughlin. 1981. 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The Journal of Cell Biology – Pubmed Central

Published: Jul 9, 2001

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