Abstract
Ubiquitin-mediated proteolysis of securin and mitotic cyclins is essential for exit from mitosis. The final step in ubiquitination of these and other proteins is catalysed by the anaphase-promoting complex (APC), a multi-subunit ubiquitin-protein ligase (E3). Little is known about the molecular reaction resulting in APC-dependent substrate ubiquitination or the role of individual APC subunits in the reaction. Using a well-defined in vitro system, we show that highly purified APC from Saccharomyces cerevisiae ubiquitinates a model cyclin substrate in a processive manner. Analysis of mutant APC lacking the Doc1/Apc10 subunit (APCdoc1Δ) indicates that Doc1 is required for processivity. The specific molecular defect in APCdoc1Δ is identified by a large increase in apparent KM for the cyclin substrate relative to the wild-type enzyme. This suggests that Doc1 stimulates processivity by limiting substrate dissociation. Addition of recombinant Doc1 to APCdoc1Δ fully restores enzyme function. Doc1-related domains are found in mechanistically distinct ubiquitin-ligase enzymes and may generally stimulate ubiquitination by contributing to substrate–enzyme affinity.
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Acknowledgements
We thank A. Szidon, A. Rudner, S. Jaspersen and E. O'Shea for reagents. We also thank members of the Morgan lab for comments on the manuscript. This work was supported a grant from the National Institute of General Medical Sciences (GM53270) to D.O.M. and by a predoctoral fellowship from the National Science Foundation to C.W.C.
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Carroll, C., Morgan, D. The Doc1 subunit is a processivity factor for the anaphase-promoting complex. Nat Cell Biol 4, 880–887 (2002). https://doi.org/10.1038/ncb871
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DOI: https://doi.org/10.1038/ncb871
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