title: Activation of the APC/C ubiquitin ligase by enhanced E2 efficiency.
abstract: The anaphase-promoting complex/cyclosome (APC/C) is a protein-ubiquitin ligase (E3) that initiates the final events of mitosis by catalyzing the ubiquitination and proteasomal destruction of securin, cyclins, and other substrates [1, 2]. Like other members of the RING family of E3s [3, 4], the APC/C catalyzes direct ubiquitin transfer from an E2-ubiquitin conjugate (E2-Ub) to lysine residues on the protein substrate. The APC/C is activated at specific cell-cycle stages by association with an activator subunit, Cdc20 or Cdh1, which provides binding sites for specific substrate sequence motifs, or degrons. Activator might also stimulate catalytic activity [5, 6], but the underlying mechanisms are not known. Here, we dissected activator function using an artificial fusion substrate in which the N-terminal region of securin was linked to an APC/C core subunit. This fusion substrate bound tightly to the APC/C and was ubiquitinated at a low rate in the absence of activator. Ubiquitination of this substrate was stimulated by activator, due primarily to a dramatic stimulation of E2 sensitivity (Km) and catalytic rate (kcat), which together resulted in a 670-fold stimulation of kcat/Km. Thus, activator is not simply a substrate adaptor, but also enhances catalysis by promoting a more efficient interaction with the E2-Ub. Interestingly, full E2 stimulation required activator interaction with degron motifs on the substrate. We conclude that formation of a complete APC/C-activator-substrate complex leads to a major enhancement of E2 efficiency, providing an unusual substrate-assisted catalytic mechanism that limits efficient ubiquitin transfer to specific substrates.
authors.affiliation: Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Vanessa A
authors.initials: VA
authors.lastname: Van Voorhis
authors.affiliation: Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: david.morgan@ucsf.edu.
authors.forename: David O
authors.initials: DO
authors.lastname: Morgan
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology*; D047468:Immunoprecipitation; D008938:Mitosis / Q000502:physiology*; D008958:Models, Molecular; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D044763:Ubiquitin-Conjugating Enzymes; D064173:Anaphase-Promoting Complex-Cyclosome