Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition.
The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.
Passmore
Lori A
LA
Section of Structural Biology and Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
McCormack
Elizabeth A
EA
Au
Shannon W N
SW
Paul
Angela
A
Willison
Keith R
KR
Harper
J Wade
JW
Barford
David
D
D064173:Anaphase-Promoting Complex-Cyclosome; D064197:Apc10 Subunit, Anaphase-Promoting Complex-Cyclosome; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
D064197:Apc10 Subunit, Anaphase-Promoting Complex-Cyclosome; D018797:Cell Cycle Proteins; C577456:DOC1 protein, S cerevisiae; C577468:MND2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; C577465:SWM1 protein, S cerevisiae