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Published on: September 17, 2012
Purification and assay of Mad2: a two-state inhibitor of anaphase-promoting complex/cyclosome
1Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041, USA.
Abstract:
To maintain the fidelity of chromosome inheritance, cells utilize a surveillance mechanism called the spindle checkpoint to sense improper attachment of sister chromatids to the mitotic spindle prior to chromosome segregation. The target of the spindle checkpoint is a ubiquitin ligase called the anaphase-promoting complex or cyclosome (APC/C). The spindle checkpoint protein Mad2 inhibits the activity of APC/C through direct binding to its activator Cdc20. Studies have shown that Mad2 has two distinct natively folded conformations and that the unusual two-state behavior of Mad2 plays a crucial role in checkpoint signaling. This article describes methods for the purification of the two Mad2 conformers and for the analysis of their activities in APC/C inhibition in Xenopus egg extracts.
Insights
The spindle checkpoint protein Mad2, crucial for accurate chromosome inheritance, has two forms that inhibit the anaphase-promoting complex/cyclosome (APC/C). This study details purifying these Mad2 conformers and analyzing their APC/C inhibitory activities.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- Cell division relies on the spindle checkpoint to ensure proper chromosome attachment.
- The anaphase-promoting complex/cyclosome (APC/C) is a key target of this checkpoint.
- Mad2 protein's two conformations are critical for spindle checkpoint signaling.
Purpose of the Study:
- To describe methods for purifying the two distinct Mad2 conformers.
- To analyze the APC/C inhibitory activities of these Mad2 conformers.
Main Methods:
- Purification of Mad2 conformers.
- In vitro assays using Xenopus egg extracts to assess APC/C inhibition.
Main Results:
- Successful purification of two distinct Mad2 conformers.
- Demonstration of differential activities of Mad2 conformers in inhibiting APC/C.
Conclusions:
- The two-state behavior of Mad2 is essential for its role in the spindle checkpoint.
- Purified Mad2 conformers can be used to study APC/C regulation in vitro.
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