Changes in the localization of the Saccharomyces cerevisiae anaphase-promoting complex upon microtubule depolymerization and spindle checkpoint activation.
The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase in the ubiquitin-mediated proteolysis pathway (UMP). To understand how the APC/C was targeted to its substrates, we performed a detailed analysis of one of the APC/C components, Cdc23p. In live cells, Cdc23-GFP localized to punctate nuclear spots surrounded by homogenous nuclear signal throughout the cell cycle. These punctate spots colocalized with two outer kinetochore proteins, Slk19p and Okp1p, but not with the spindle pole body protein, Spc42p. In late anaphase, the Cdc23-GFP was also visualized along the length of the mitotic spindle. We hypothesized that spindle checkpoint activation may affect the APC/C nuclear spot localization. Localization of Cdc23-GFP was disrupted upon nocodazole treatment in the kinetochore mutant okp1-5 and in the cdc20-1 mutant. Cdc23-GFP nuclear spot localization was not affected in the ndc10-1 mutant, which is defective in spindle checkpoint function. Additional studies using a mad2Delta strain revealed a microtubule dependency of Cdc23-GFP spot localization, whether or not the checkpoint response was activated. On the basis of these data, we conclude that Cdc23p localization was dependent on microtubules and was affected by specific types of kinetochore disruption.
Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. pmelloy@molbio.princeton.edu
Patricia G
PG
Melloy
Sandra L
SL
Holloway
D064173:Anaphase-Promoting Complex-Cyclosome; D064196:Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D018386:Kinetochores / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D015739:Nocodazole; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism; D013696:Temperature; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics*
D064196:Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome; C577441:CDC23 protein, S cerevisiae; D018797:Cell Cycle Proteins; D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D015739:Nocodazole