Limits...
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).

Show MeSH

Related in: MedlinePlus

APCCdh1 activity is inactivated during S phase. (A) Constitutive 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 α-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 α-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 clb5Δ strains expressing C254 under the control of the GALL promoter were grown in galactose-containing medium, arrested in α-factor, released, and samples collected as in B.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196868&req=5

fig3: APCCdh1 activity is inactivated during S phase. (A) Constitutive 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 α-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 α-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 clb5Δ strains expressing C254 under the control of the GALL promoter were grown in galactose-containing medium, arrested in α-factor, released, and samples collected as in B.

Mentions: The finding that Ase1p was degraded by APCCdh1 at the cdc4 block suggested that G1 CDK activity was not sufficient to fully inactivate APCCdh1. To monitor APCCdh1 activity 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 throughout the cell cycle when cells are grown in galactose-containing medium. Importantly, constitutive expression of C254 did not affect cell growth (data not shown) or cell cycle progression (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.


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 activity is inactivated during S phase. (A) Constitutive 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 α-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 α-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 clb5Δ strains expressing C254 under the control of the GALL promoter were grown in galactose-containing medium, arrested in α-factor, released, and samples collected as in B.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: APCCdh1 activity is inactivated during S phase. (A) Constitutive 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 α-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 α-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 clb5Δ strains expressing C254 under the control of the GALL promoter were grown in galactose-containing medium, arrested in α-factor, released, and samples collected as in B.
Mentions: The finding that Ase1p was degraded by APCCdh1 at the cdc4 block suggested that G1 CDK activity was not sufficient to fully inactivate APCCdh1. To monitor APCCdh1 activity 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 throughout the cell cycle when cells are grown in galactose-containing medium. Importantly, constitutive expression of C254 did not affect cell growth (data not shown) or cell cycle progression (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.

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