The Anaphase Promoting Complex or Cyclosome (APC/C) is critical to the control of mitosis. The APC/C is an ubiquitin ligase that targets specific mitotic regulators for proteolysis at distinct times in mitosis, but how this is achieved is not well understood. We have addressed this question by determining whether the same substrate, cyclin B1, is recognised in the same way by the APC/C at different times in mitosis. Unexpectedly, we find that distinct but overlapping motifs in cyclin B1 are recognised by the APC/C in metaphase compared with anaphase, and this does not depend on the exchange of Cdc20 for Cdh1. Thus, changes in APC/C substrate specificity in mitosis can potentially be conferred by altering interaction sites in addition to exchanging Cdc20 for Cdh1.
The budding yeast proteins Dma1 and Dma2 are members of the unique FHA-RING domain protein family and are linked to mitotic regulation and septin organization by ill-defined mechanisms. We show that Dma2 has ubiquitin ligase activity, and that septins Shs1 and Cdc11 are likely direct in vivo targets. We further propose that human RNF8, rather than Chfr, is the mammalian Dma homolog. As in yeast, RNF8 localizes to the centrosomes and cell division sites and promotes ubiquitylation of the septin SEPT7, whose depletion increases cell division anomalies. Together, these findings reveal evolutionary and functional conservation of Dma proteins, and suggest that RNF8 maintains genome stability through independent, yet analogous, nuclear and cytoplasmic ubiquitylation activities.
We conducted a mitotic localization study on gene products encoded by 56 uncharacterized fission yeast ORFs that were transcriptionally up-regulated during meiotic division. Despite meiotic gene induction, these genes were expressed during mitosis as well. Seven gene products were localized in the nucleus and/or chromatin; another one was a mitosis-specific spindle pole body component and, intriguingly, its human homologue was also localized in the centrosome of cultured HeLa cells. Two products appeared to be localized in cytoplasmic microtubules, whereas four were mitochondrial proteins. Three other proteins were found in the medial ring upon cytokinesis and another was localized on the entire cell periphery. The remaining 38 proteins were detected in the cytoplasm and showed varied spatial patterns. This systematic study helps our integrated understanding of all the protein functions in the fission yeast as a eukaryotic model.
The anaphase-promoting complex (APC/C), a ubiquitin ligase, is the target of the spindle-assembly checkpoint (SAC), and it ubiquitylates protein substrates whose degradation regulates progress through mitosis. The identity of the ubiquitin-conjugating (E2) enzymes that work with the APC/C is unclear. In an RNA interference (RNAi) screen for factors that modify release from drug-induced SAC activation, we identified the E2 enzyme UBE2S as an APC/C auxiliary factor that promotes mitotic exit. UBE2S is dispensable in a normal mitosis, but its depletion prolongs drug-induced mitotic arrest and suppresses mitotic slippage. In vitro, UBE2S elongates ubiquitin chains initiated by the E2 enzymes UBCH10 and UBCH5, enhancing the degradation of APC/C substrates by the proteasome. Indeed, following release from SAC-induced mitotic arrest, UBE2S-depleted cells neither degrade crucial APC/C substrates, nor silence this checkpoint, whereas bypassing the SAC through BUBR1 depletion or Aurora-B inhibition negates the requirement for UBE2S. Thus, UBE2S functions with the APC/C in a two-step mechanism to control substrate ubiquitylation that is essential for mitotic exit after prolonged SAC activation, providing a new model for APC/C function in human cells.
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