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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 regulation of Cdc20p expression. (A) Cdc20p is an APCCdh1 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 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ strains as for Ase1p in the legend to Fig. 1 B. (B) Expression of stable Cdc20p in G1 delays S phase progression. Wild-type strains containing either vector alone or GAL1,10::Cdc20ΔBox1+2-HA (Prinz et al., 1998) were arrested with α-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) Inability to degrade Cdc20p in G1 delays S phase progression. Wild-type strains containing either GAL1,10::Cdc20-HA or GAL1,10::Cdc20ΔBox1+2-HA (Prinz et al., 1998) were arrested with α-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 Cdc20ΔBox1+2 were determined by Western blotting and quantified as described in Materials and methods.
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fig5: APCCdh1 regulation of Cdc20p expression. (A) Cdc20p is an APCCdh1 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 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ strains as for Ase1p in the legend to Fig. 1 B. (B) Expression of stable Cdc20p in G1 delays S phase progression. Wild-type strains containing either vector alone or GAL1,10::Cdc20ΔBox1+2-HA (Prinz et al., 1998) were arrested with α-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) Inability to degrade Cdc20p in G1 delays S phase progression. Wild-type strains containing either GAL1,10::Cdc20-HA or GAL1,10::Cdc20ΔBox1+2-HA (Prinz et al., 1998) were arrested with α-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 Cdc20ΔBox1+2 were determined by Western blotting and quantified as described in Materials and methods.

Mentions: The persistence of APCCdh1 activity through late G1 suggested that the degradation of certain APCCdh1 substrates might be required for the normal execution of S phase. One appealing candidate for such a substrate is the APC activator Cdc20p. Metazoan Cdc20p is an APCCdh1 substrate (Pfleger and Kirschner, 2000; Sorensen et al., 2000), and premature activation of APCCdc20 might interfere with the expression of S phase cyclins. Indeed, we found that degradation of yeast Cdc20p is APCCdh1 dependent in cdc4-arrested cells (Fig. 5 A).


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 regulation of Cdc20p expression. (A) Cdc20p is an APCCdh1 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 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ strains as for Ase1p in the legend to Fig. 1 B. (B) Expression of stable Cdc20p in G1 delays S phase progression. Wild-type strains containing either vector alone or GAL1,10::Cdc20ΔBox1+2-HA (Prinz et al., 1998) were arrested with α-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) Inability to degrade Cdc20p in G1 delays S phase progression. Wild-type strains containing either GAL1,10::Cdc20-HA or GAL1,10::Cdc20ΔBox1+2-HA (Prinz et al., 1998) were arrested with α-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 Cdc20ΔBox1+2 were determined by Western blotting and quantified as described in Materials and methods.
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fig5: APCCdh1 regulation of Cdc20p expression. (A) Cdc20p is an APCCdh1 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 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ strains as for Ase1p in the legend to Fig. 1 B. (B) Expression of stable Cdc20p in G1 delays S phase progression. Wild-type strains containing either vector alone or GAL1,10::Cdc20ΔBox1+2-HA (Prinz et al., 1998) were arrested with α-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) Inability to degrade Cdc20p in G1 delays S phase progression. Wild-type strains containing either GAL1,10::Cdc20-HA or GAL1,10::Cdc20ΔBox1+2-HA (Prinz et al., 1998) were arrested with α-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 Cdc20ΔBox1+2 were determined by Western blotting and quantified as described in Materials and methods.
Mentions: The persistence of APCCdh1 activity through late G1 suggested that the degradation of certain APCCdh1 substrates might be required for the normal execution of S phase. One appealing candidate for such a substrate is the APC activator Cdc20p. Metazoan Cdc20p is an APCCdh1 substrate (Pfleger and Kirschner, 2000; Sorensen et al., 2000), and premature activation of APCCdc20 might interfere with the expression of S phase cyclins. Indeed, we found that degradation of yeast Cdc20p is APCCdh1 dependent in cdc4-arrested cells (Fig. 5 A).

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