The Doc1 subunit is a processivity factor for the anaphase-promoting complex.
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 (APC(doc1 Delta)) indicates that Doc1 is required for processivity. The specific molecular defect in APC(doc1 Delta) is identified by a large increase in apparent K(M) 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 APC(doc1 Delta) 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.
Carroll
Christopher W
CW
Department of Physiology, University of California, San Francisco, CA 94143-0444, USA.
Morgan
David O
DO
D000705:Anaphase; D064173:Anaphase-Promoting Complex-Cyclosome; D064197:Apc10 Subunit, Anaphase-Promoting Complex-Cyclosome; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D013379:Substrate Specificity; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000502:physiology*
D064197:Apc10 Subunit, Anaphase-Promoting Complex-Cyclosome; D018797:Cell Cycle Proteins; C577456:DOC1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases