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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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Ase1p domain sufficient for APCCdh1-mediated degradation. (A) Stability of Ase1p NH2-terminal truncations fused to GST in α-factor–arrested cells. Chimeras were expressed from the GALL promoter for 30 min in α-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 α-factor, shifted to 36°C 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 determined. (C) cdc4 cells containing C254 or a db mutant of C254 (C254-db) were arrested at 36°C, and the half-lives of the fusion proteins were determined 30 min after the addition of galactose. (D) The half-life of C254 was determined in arrested cdc4 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ cells. (E) Cells constitutively expressing C254 (the GST–R632-I885 fusion protein) were prepared for immunofluorescence. No signal was detected in this strain grown under repressing conditions (glucose; data not shown). C254 is diffusely localized to the nucleus and appears to be at higher levels in mitotic cells.
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fig2: Ase1p domain sufficient for APCCdh1-mediated degradation. (A) Stability of Ase1p NH2-terminal truncations fused to GST in α-factor–arrested cells. Chimeras were expressed from the GALL promoter for 30 min in α-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 α-factor, shifted to 36°C 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 determined. (C) cdc4 cells containing C254 or a db mutant of C254 (C254-db) were arrested at 36°C, and the half-lives of the fusion proteins were determined 30 min after the addition of galactose. (D) The half-life of C254 was determined in arrested cdc4 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ cells. (E) Cells constitutively expressing C254 (the GST–R632-I885 fusion protein) were prepared for immunofluorescence. No signal was detected in this strain grown under repressing conditions (glucose; data not shown). C254 is diffusely localized to the nucleus and appears to be at higher levels in mitotic cells.

Mentions: Ase1p is a microtubule-binding and cross-linking protein that is required for the structural integrity of the anaphase spindle (Juang et al., 1997; unpublished data). Although Ase1p is not expected to regulate APC/C, high levels of Ase1p activate the spindle assembly checkpoint and thereby might indirectly affect APC/C activity (Juang et al., 1997; for review see Amon, 1999). To create an inert reporter of APCCdh1 activity, we constructed chimeras between Ase1p and glutathione S-transferase (GST). These chimeras enabled us to define the minimal Ase1p sequence necessary for APCCdh1-mediated degradation. The half-lives of the GST–Ase1p fusions were measured in α-factor–arrested cells (Fig. 2 A). A chimera containing the COOH-terminal 254 amino acids (R632-I885) of Ase1p was degraded with similar kinetics to full-length Ase1p. Further deletions up to amino acid 802 from the COOH-terminal end of R632-I885 were also rapidly degraded in α-factor–arrested cells. By contrast, deletion of 22 amino acids on the NH2-terminal side of the R632-I885 peptide resulted in a stable chimera (T654-I885; Fig. 2 A). 7 of these 22 residues are lysines, raising the possibility that these may be the site(s) of Ase1p ubiquitination. Together, these results suggest that residues 632–802 comprise the minimal sequence for Ase1p degradation in α-factor–arrested cells.


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)

Ase1p domain sufficient for APCCdh1-mediated degradation. (A) Stability of Ase1p NH2-terminal truncations fused to GST in α-factor–arrested cells. Chimeras were expressed from the GALL promoter for 30 min in α-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 α-factor, shifted to 36°C 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 determined. (C) cdc4 cells containing C254 or a db mutant of C254 (C254-db) were arrested at 36°C, and the half-lives of the fusion proteins were determined 30 min after the addition of galactose. (D) The half-life of C254 was determined in arrested cdc4 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ cells. (E) Cells constitutively expressing C254 (the GST–R632-I885 fusion protein) were prepared for immunofluorescence. No signal was detected in this strain grown under repressing conditions (glucose; data not shown). C254 is diffusely localized to the nucleus and appears to be at higher levels in mitotic cells.
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Related In: Results  -  Collection

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

fig2: Ase1p domain sufficient for APCCdh1-mediated degradation. (A) Stability of Ase1p NH2-terminal truncations fused to GST in α-factor–arrested cells. Chimeras were expressed from the GALL promoter for 30 min in α-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 α-factor, shifted to 36°C 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 determined. (C) cdc4 cells containing C254 or a db mutant of C254 (C254-db) were arrested at 36°C, and the half-lives of the fusion proteins were determined 30 min after the addition of galactose. (D) The half-life of C254 was determined in arrested cdc4 cdh1Δ clb6Δ and cdc4 CDH1 clb6Δ cells. (E) Cells constitutively expressing C254 (the GST–R632-I885 fusion protein) were prepared for immunofluorescence. No signal was detected in this strain grown under repressing conditions (glucose; data not shown). C254 is diffusely localized to the nucleus and appears to be at higher levels in mitotic cells.
Mentions: Ase1p is a microtubule-binding and cross-linking protein that is required for the structural integrity of the anaphase spindle (Juang et al., 1997; unpublished data). Although Ase1p is not expected to regulate APC/C, high levels of Ase1p activate the spindle assembly checkpoint and thereby might indirectly affect APC/C activity (Juang et al., 1997; for review see Amon, 1999). To create an inert reporter of APCCdh1 activity, we constructed chimeras between Ase1p and glutathione S-transferase (GST). These chimeras enabled us to define the minimal Ase1p sequence necessary for APCCdh1-mediated degradation. The half-lives of the GST–Ase1p fusions were measured in α-factor–arrested cells (Fig. 2 A). A chimera containing the COOH-terminal 254 amino acids (R632-I885) of Ase1p was degraded with similar kinetics to full-length Ase1p. Further deletions up to amino acid 802 from the COOH-terminal end of R632-I885 were also rapidly degraded in α-factor–arrested cells. By contrast, deletion of 22 amino acids on the NH2-terminal side of the R632-I885 peptide resulted in a stable chimera (T654-I885; Fig. 2 A). 7 of these 22 residues are lysines, raising the possibility that these may be the site(s) of Ase1p ubiquitination. Together, these results suggest that residues 632–802 comprise the minimal sequence for Ase1p degradation in α-factor–arrested cells.

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