The dephosphorylated form of the anaphase-promoting complex protein Cdc27/Apc3 concentrates on kinetochores and chromosome arms in mitosis.
Cell cycle regulated protein ubiquitination and degradation within subcellular domains may be essential for the normal progression of mitosis. Cdc27 is a conserved component of an essential M-phase ubiquitin-protein ligase called the anaphase-promoting complex/cyclosome. We examined the subcellular distribution of Cdc27 in greater detail in mammalian cells and found Cdc27 concentrated at spindle poles and on spindle microtubules as previously described, but also found Cdc27 at kinetochores and along chromosome arms. This localization was not dependent on intact microtubules. While the great majority of Cdc27 protein in M phase cells is highly phosphorylated, only the dephosphorylated form of Cdc27 was found associated with isolated chromosomes. Kinases that also associate with isolated chromosomes catalyzed the in vitro phosphorylation of the chromosome-associated Cdc27. Microinjection of anti-Cdc27 antibody into cells causes arrest at metaphase. Microinjection of cells with anti-Mad2 antibody normally induces premature anaphase onset resulting in catastrophic nondisjunction of the chromosomes. However, coinjection of anti-Cdc27 antibody with anti-Mad2 antibody resulted in metaphase arrest. The association of dephosphorylated APC/C components with mitotic chromosomes suggests mechanisms by which the spindle checkpoint may regulate APC/C activity at mitosis.
Topper
Leana M
LM
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.
Campbell
Michael S
MS
Tugendreich
Stuart
S
Daum
John R
JR
Burke
Daniel J
DJ
Hieter
Philip
P
Gorbsky
Gary J
GJ
D000818:Animals; D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002875:Chromosomes / Q000648:ultrastructure*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D018386:Kinetochores / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008938:Mitosis; D010766:Phosphorylation; D011418:Prophase; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases
D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; C577495:CDC20 protein, S cerevisiae; C577355:CDC27 protein, S cerevisiae; C577366:CDC27 protein, human; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases