Closed MAD2 (C-MAD2) is selectively incorporated into the mitotic checkpoint complex (MCC).
The mitotic checkpoint is a specialized signal transduction pathway that monitors kinetochore-microtubule attachment to achieve faithful chromosome segregation. MAD2 is an evolutionarily conserved mitotic checkpoint protein that exists in open (O) and closed (C) conformations. The increase of intracellular C-MAD2 level during mitosis, through O→C-MAD2 conversion as catalyzed by unattached kinetochores, is a critical signaling event for the mitotic checkpoint. However, it remains controversial whether MAD2 is an integral component of the effector of the mitotic checkpoint--the Mitotic Checkpoint Complex (MCC). We show here that endogenous human MCC is assembled by first forming a BUBR1:BUB3:CDC20 complex in G2 and then selectively incorporating C-MAD2 during mitosis. Nevertheless, MCC can be induced to form in G1/S cells by expressing a C-conformation locked MAD2 mutant, indicating intracellular level of C-MAD2 as a major limiting factor for MCC assembly. In addition, a recombinant MCC containing C-MAD2 exhibits effective inhibitory activity towards APC/C isolated from mitotic HeLa cells, while a recombinant BUBR1:BUB3:CDC20 ternary complex is ineffective at comparable concentrations despite association with APC/C. These results help establish a direct connection between a major signal transducer (C-MAD2) and the potent effector (MCC) of the mitotic checkpoint, and provide novel insights into protein-protein interactions during assembly of a functional MCC.
Department of Biological Sciences, University of Toledo, Toledo, OH, USA.
Aaron R
AR
Tipton
Michael
M
Tipton
Tim
T
Yen
Song-Tao
ST
Liu
D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints; D064246:Mad2 Proteins; D008938:Mitosis; D046912:Multiprotein Complexes; D000075223:Poly-ADP-Ribose Binding Proteins; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
C489444:BUB3 protein, human; D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D046912:Multiprotein Complexes; D000075223:Poly-ADP-Ribose Binding Proteins; D012097:Repressor Proteins; C577488:CDC20 protein, human; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases