Recruitment of Cdc20 to the kinetochore requires BubR1 but not Mad2 in Drosophila melanogaster.
To prevent aneuploidy, cells require a mitotic surveillance mechanism, the spindle assembly checkpoint (SAC). The SAC prevents metaphase/anaphase transition by blocking the ubiquitylation and destruction of cyclin B and securin via the Cdc20-activated anaphase-promoting complex or cyclosome (APC/C)-mediated proteolysis pathway. This checkpoint involves the kinetochore proteins Mad2, BubR1, and Cdc20. Mad2 and BubR1 are inhibitors of the APC/C, but Cdc20 is an activator. Exactly how the SAC regulates Cdc20 via unattached kinetochores remains unclear; in vertebrates, most current models suggest that kinetochore-bound Mad2 is required for initial binding to Cdc20 to form a stable complex that includes BubR1. Here, we show that the Mad2 kinetochore dimerization recruitment mechanism is conserved and that the recruitment of Cdc20 to kinetochores in Drosophila requires BubR1 but not Mad2. BubR1 and Mad2 can bind to Cdc20 independently, and the interactions are enhanced after cells are arrested at mitosis by the depletion of Cdc27 using RNA interference (RNAi) in S2 cells or by MG132 treatment in syncytial embryos. These findings offer an explanation of why BubR1 is more important than Mad2 for SAC function in flies. These findings could lead to a better understanding of vertebrate SAC mechanisms.
Li
Deyu
D
Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
Morley
Gary
G
Whitaker
Michael
M
Huang
Jun-Yong
JY
D000595:Amino Acid Sequence; D000818:Animals; D064199:Cdc20 Proteins; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D003078:Colchicine / Q000494:pharmacology; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000033:anatomy & histology / Q000187:drug effects / Q000502:physiology; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D064246:Mad2 Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D008941:Spindle Apparatus / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology
C506005:BubR1 protein, Drosophila; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D064246:Mad2 Proteins; D011993:Recombinant Fusion Proteins; D050257:Tubulin Modulators; C577502:fzy protein, Drosophila; D003078:Colchicine