Related Experiment Video
Updated: Jun 22, 2026
08:13
Manipulation and Analysis of Cell Cycle-Dependent Processes in Budding Yeast
Published on: September 26, 2025
An in vitro assay for Cdc20-dependent mitotic anaphase-promoting complex activity from budding yeast
Scott C Schuyler1, Andrew W Murray
1Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|May 29, 2009
Summary
This study details a quantitative in vitro assay for the Anaphase-Promoting Complex (APC), crucial for cell cycle control. The assay uses a highly active form of APC (APC(Cdc20)) from yeast to study cell division regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell cycle progression relies on regulated protein degradation via the ubiquitin-proteasome system.
- The Anaphase-Promoting Complex (APC), an E3 ubiquitin ligase, governs key cell cycle transitions, notably the metaphase-to-anaphase switch.
- Cdc20 acts as a critical co-factor, activating the APC (APC(Cdc20)) for substrate ubiquitination.
Purpose of the Study:
- To establish a robust, quantitative in vitro assay for studying the mitotic Anaphase-Promoting Complex (APC).
- To enable detailed investigation of APC enzymology, regulation, and substrate identification.
- To leverage the well-characterized budding yeast Saccharomyces cerevisiae for assay development.
Main Methods:
- Purification of a highly active form of the Anaphase-Promoting Complex (APC) from mitotic yeast cells.
- Development of a quantitative in vitro assay system for APC(Cdc20) activity.
- Utilizing budding yeast (Saccharomyces cerevisiae) as the source of purified proteins.
Main Results:
- A reliable quantitative in vitro assay for mitotic APC(Cdc20) was successfully established.
- The assay facilitates the identification and validation of APC target substrates.
- The system supports in-depth studies of APC enzymology and regulatory mechanisms.
Conclusions:
- The developed quantitative in vitro APC(Cdc20) assay provides a powerful tool for cell cycle research.
- This assay enhances the study of ubiquitin-dependent proteolysis in mitosis.
- Findings contribute to understanding cell cycle control mechanisms using a model organism system.
More Related Videos
07:48Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae
Published on: October 11, 2022
13:59Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos
Published on: June 14, 2012
Related Concept Videos
00:50Anaphase Promoting Complex
The stepwise destruction of specific proteins is necessary for the progression and completion of the cell cycle. Such proteins are ubiquitinated by ubiquitin ligases and then subsequently destroyed by the proteasome. The SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC) are two important ubiquitin ligases involved in cell cycle progression. While SCF is active throughout the cell cycle, APC gets activated during metaphase to anaphase transition. Cdc20 or Cdh1 binds to APC and...
02:17Separation of Sister Chromatids
At the transition from prophase to metaphase, there is a reduction in cohesion along the chromosomal arms, resulting in the resolution of sister chromatids. However, residual cohesin connections remain to hold the sister chromatids together until the transition from metaphase to anaphase. The residual connection prevents any premature separation of sister chromatids, blocking the risks of aneuploidy within the daughter cells.
At the onset of anaphase, separase, a proteolytic enzyme, is...
At the onset of anaphase, separase, a proteolytic enzyme, is...
02:19The Spindle Assembly Checkpoint
The spindle assembly checkpoint is a molecular surveillance mechanism ensuring the fidelity of chromosome segregation during anaphase. The checkpoint monitors the completion of all the prerequisite steps before chromosome segregation to determine whether the segregation process should proceed or be delayed.
Many proteins function together to control the spindle assembly checkpoint. Mutations affecting these proteins may allow cells to proceed into anaphase prematurely, resulting in the...
Many proteins function together to control the spindle assembly checkpoint. Mutations affecting these proteins may allow cells to proceed into anaphase prematurely, resulting in the...
02:15M-Cdk Drives Transition Into Mitosis
Checkpoints throughout the cell cycle serve as safeguards and gatekeepers, allowing the cell cycle to progress in favorable conditions and slow or halt it in problematic ones. This regulation is known as the cell cycle control system.
Cyclin-dependent kinases, or Cdks, work in concert with cyclins to control cell cycle transitions. M-Cdk, a complex of Cdk1 bound to M cyclin, is a well-known example of this coordinated control that drives the transition from the G2 to the M phase.
M cyclin...
Cyclin-dependent kinases, or Cdks, work in concert with cyclins to control cell cycle transitions. M-Cdk, a complex of Cdk1 bound to M cyclin, is a well-known example of this coordinated control that drives the transition from the G2 to the M phase.
M cyclin...
02:34Inhibition of Cdk Activity
The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex...