Identification of novel anaphase -promoting complex (APC/C) interactions and regulation of APC/C localization at the kinetochore in budding yeast

The APC/C is a ubiquitin ligase (E3) that targets proteins for cell cycle-regulated proteolysis through ubiquitination. Cdc23p, an APC/C subunit, directly binds the mitotic cyclin, Clb2p, and associates with the kinetochore protein Okp1p by two-hybrid analysis. In these studies, the APC/C has been localized in budding yeast by tagging two APC/C subunits, CDC23 (APC8) and APC9 with green fluorescent protein (GFP). Microscopic analysis of CDC23-GFP and APC9-GFP indicates that the APC/C is nuclear throughout the cell cycle. In addition, the complex is targeted to small nuclear spots co-localizing with two kinetochore proteins, Slk19p and Okp1p. The APC/C also co-localizes with the mitotic anaphase spindle. Regulation of APC/C localization may be one mechanism for controlling APC/C activity. To test this model, APC/C kinetochore localization has been monitored after spindle checkpoint activation. This checkpoint can halt the cell cycle upon spindle damage by inactivating the APC/C. APC/C kinetochore localization is disrupted upon checkpoint activation in an okp1-5 kinetochore mutant and after nocodazole treatment. However, spindle checkpoint activation by overexpression of MPS1 does not cause a loss of the sub-nuclear localization of the APC/C. In the ndc10-1 kinetochore mutant, which lacks a checkpoint sensor, the APC/C localization is unchanged. Therefore, in the case of defective microtubule-kinetochore attachment and/or tension, spindle checkpoint activation results in displacement of the APC/C from the kinetochore, a possible mechanism of APC/C inactivation. In addition, these studies indicate that the APC/C regulator Cdc20p plays a role in maintaining the APC/C at the kinetochore