Purification and assay of Mad2: a two-state inhibitor of anaphase-promoting complex/cyclosome.
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.
Luo
Xuelian
X
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041, USA.
Yu
Hongtao
H
D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D003001:Cloning, Molecular / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli; D006801:Humans; D064246:Mad2 Proteins; D009682:Magnetic Resonance Spectroscopy; D010063:Ovum; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D043743:Ubiquitin-Protein Ligase Complexes / Q000037:antagonists & inhibitors*; D014982:Xenopus laevis
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D012097:Repressor Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome