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Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p.

Huang JN, Park I, Ellingson E, Littlepage LE, Pellman D - J. Cell Biol. (2001)

Bottom Line: Complete inactivation of APC(Cdh1) requires S phase cyclins.Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression.This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, The Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA.

ABSTRACT
Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).

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APCCdh1 is active in late G1. (A) cdc4 strains containing GAL1,10::ASE1 or GAL1,10::ASE1-DB (Juang et al., 1997) were arrested in yeast extract and peptone (YEP) raffinose at 36°C. Expression from the GAL1,10 promoter was induced for 60 min, and the half-life of Ase1p or Ase1p-db was determined. G1 arrest was confirmed by FACS®. (B) The half-life of Ase1p was determined in arrested cdc4 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ strains as above. The clb6Δ mutation was introduced into these strains to promote efficient arrest. cdc4 cdh1Δ double mutants arrest poorly in late G1, probably because of persistent low levels of B-type cyclins from the previous mitosis (unpublished data). (C) cdc4 strains containing an epitope-tagged APC/C subunit (Cdc16p-myc6), or the untagged control, and HA-tagged Cdh1p (HA3-Cdh1p) were arrested at 36°C, and APC/C was immunoprecipitated using a monoclonal antibody directed against the myc epitope. Extracts and immunoprecipitates were probed with 12CA5 to detect HA3-Cdh1p.
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fig1: APCCdh1 is active in late G1. (A) cdc4 strains containing GAL1,10::ASE1 or GAL1,10::ASE1-DB (Juang et al., 1997) were arrested in yeast extract and peptone (YEP) raffinose at 36°C. Expression from the GAL1,10 promoter was induced for 60 min, and the half-life of Ase1p or Ase1p-db was determined. G1 arrest was confirmed by FACS®. (B) The half-life of Ase1p was determined in arrested cdc4 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ strains as above. The clb6Δ mutation was introduced into these strains to promote efficient arrest. cdc4 cdh1Δ double mutants arrest poorly in late G1, probably because of persistent low levels of B-type cyclins from the previous mitosis (unpublished data). (C) cdc4 strains containing an epitope-tagged APC/C subunit (Cdc16p-myc6), or the untagged control, and HA-tagged Cdh1p (HA3-Cdh1p) were arrested at 36°C, and APC/C was immunoprecipitated using a monoclonal antibody directed against the myc epitope. Extracts and immunoprecipitates were probed with 12CA5 to detect HA3-Cdh1p.

Mentions: To determine if Ase1p destruction was inactivated by G1 CDK activity, the half-life of Ase1p was determined in a cdc4 mutant, which arrests before S phase with high G1 CDK activity and high levels of Sic1p. We found that Ase1p is rapidly degraded at the cdc4 block with similar kinetics to that observed previously in α-factor–arrested cells (Fig. 1 A; Juang et al., 1997). The 5–10 min half-life of Ase1p at the cdc4 arrest point contrasts sharply with the >60 min half-life of Ase1p observed in cycling or nocodazole-arrested cells (Juang et al., 1997; data not shown). Ase1p is also rapidly degraded in a skp1-11 strain, which also arrests in late G1 (data not shown). This also contrasts with the degradation of the APCCdh1 substrate Clb2p, which is stable at the cdc4 block (Amon et al., 1994; Amon, 1997; unpublished data; see Fig. 4 B, glu). Like Ase1p degradation in α-factor–arrested cells, Ase1p degradation at the cdc4 block required Cdh1p and the destruction box (db), a cis-acting sequence for APC/C-mediated degradation (Fig. 1, A and B). Therefore, Ase1p is rapidly degraded by APCCdh1 in the presence of high levels of G1 CDK activity.


Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p.

Huang JN, Park I, Ellingson E, Littlepage LE, Pellman D - J. Cell Biol. (2001)

APCCdh1 is active in late G1. (A) cdc4 strains containing GAL1,10::ASE1 or GAL1,10::ASE1-DB (Juang et al., 1997) were arrested in yeast extract and peptone (YEP) raffinose at 36°C. Expression from the GAL1,10 promoter was induced for 60 min, and the half-life of Ase1p or Ase1p-db was determined. G1 arrest was confirmed by FACS®. (B) The half-life of Ase1p was determined in arrested cdc4 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ strains as above. The clb6Δ mutation was introduced into these strains to promote efficient arrest. cdc4 cdh1Δ double mutants arrest poorly in late G1, probably because of persistent low levels of B-type cyclins from the previous mitosis (unpublished data). (C) cdc4 strains containing an epitope-tagged APC/C subunit (Cdc16p-myc6), or the untagged control, and HA-tagged Cdh1p (HA3-Cdh1p) were arrested at 36°C, and APC/C was immunoprecipitated using a monoclonal antibody directed against the myc epitope. Extracts and immunoprecipitates were probed with 12CA5 to detect HA3-Cdh1p.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196868&req=5

fig1: APCCdh1 is active in late G1. (A) cdc4 strains containing GAL1,10::ASE1 or GAL1,10::ASE1-DB (Juang et al., 1997) were arrested in yeast extract and peptone (YEP) raffinose at 36°C. Expression from the GAL1,10 promoter was induced for 60 min, and the half-life of Ase1p or Ase1p-db was determined. G1 arrest was confirmed by FACS®. (B) The half-life of Ase1p was determined in arrested cdc4 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ strains as above. The clb6Δ mutation was introduced into these strains to promote efficient arrest. cdc4 cdh1Δ double mutants arrest poorly in late G1, probably because of persistent low levels of B-type cyclins from the previous mitosis (unpublished data). (C) cdc4 strains containing an epitope-tagged APC/C subunit (Cdc16p-myc6), or the untagged control, and HA-tagged Cdh1p (HA3-Cdh1p) were arrested at 36°C, and APC/C was immunoprecipitated using a monoclonal antibody directed against the myc epitope. Extracts and immunoprecipitates were probed with 12CA5 to detect HA3-Cdh1p.
Mentions: To determine if Ase1p destruction was inactivated by G1 CDK activity, the half-life of Ase1p was determined in a cdc4 mutant, which arrests before S phase with high G1 CDK activity and high levels of Sic1p. We found that Ase1p is rapidly degraded at the cdc4 block with similar kinetics to that observed previously in α-factor–arrested cells (Fig. 1 A; Juang et al., 1997). The 5–10 min half-life of Ase1p at the cdc4 arrest point contrasts sharply with the >60 min half-life of Ase1p observed in cycling or nocodazole-arrested cells (Juang et al., 1997; data not shown). Ase1p is also rapidly degraded in a skp1-11 strain, which also arrests in late G1 (data not shown). This also contrasts with the degradation of the APCCdh1 substrate Clb2p, which is stable at the cdc4 block (Amon et al., 1994; Amon, 1997; unpublished data; see Fig. 4 B, glu). Like Ase1p degradation in α-factor–arrested cells, Ase1p degradation at the cdc4 block required Cdh1p and the destruction box (db), a cis-acting sequence for APC/C-mediated degradation (Fig. 1, A and B). Therefore, Ase1p is rapidly degraded by APCCdh1 in the presence of high levels of G1 CDK activity.

Bottom Line: Complete inactivation of APC(Cdh1) requires S phase cyclins.Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression.This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, The Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA.

ABSTRACT
Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).

Show MeSH
Related in: MedlinePlus