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Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations
Published on: September 17, 2012
Cell cycle -dependent proteolysis in plants. Identification Of the destruction box pathway and metaphase arrest
1Institut de Biologie Moleculaire des Plantes du CNRS, 12 rue du General Zimmer, 67084 Strasbourg Cedex, France.
Abstract:
It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable.
Insights
Plant mitotic cyclins undergo cell cycle-specific proteolysis, regulated by their destruction box motif. This post-translational control is crucial for cell cycle progression and exit from mitosis.
Area of Science:
- Plant molecular biology
- Cell cycle regulation
- Protein degradation
Background:
- Mitotic cyclins are essential regulators of the cell cycle.
- Rapid degradation of cyclins during anaphase inactivates Cdc2 and promotes mitotic exit.
- Proteolysis is mediated by the ubiquitin/26S proteasome system and requires a destruction box motif.
Purpose of the Study:
- To investigate the stability and degradation of mitotic cyclins in plants.
- To determine if plant cyclins are subject to cell cycle-specific proteolysis.
- To identify the role of the destruction box motif in plant cyclin degradation.
Main Methods:
- Construction and expression of cyclin-chloramphenicol acetyltransferase (CAT) fusion proteins in tobacco BY2 cells.
- Analysis of fusion protein stability and cell cycle-specific degradation.
- Site-directed mutagenesis of the destruction box motif.
- Treatment with the proteasome inhibitor MG132.
Main Results:
- N-terminal domains of tobacco cyclins A and B fused to CAT conferred cell cycle-specific oscillation and degradation.
- Mutations in the destruction box abolished cell cycle-specific proteolysis.
- Cyclin A-CAT and cyclin B-CAT were degraded post-metaphase, with distinct degradation timing.
- Proteasome inhibition by MG132 caused reversible metaphase arrest, stabilizing both fusion proteins.
Conclusions:
- Mitotic cyclins in plants are regulated by post-translational proteolysis.
- The destruction box motif is essential for cell cycle-specific degradation of plant cyclins.
- Proteasome-mediated degradation is a key mechanism controlling plant cell cycle progression.
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