Analysis of activator-binding sites on the APC/C supports a cooperative substrate-binding mechanism.
The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase essential for the completion of mitosis in all eukaryotic cells. Substrates are recruited to the APC/C by activator proteins (Cdc20 or Cdh1), but it is not known where substrates are bound during catalysis. We explored this problem by analyzing mutations in the tetratricopeptide-repeat-containing APC/C subunits. We identified residues in Cdc23 and Cdc27 that are required for APC/C binding to Cdc20 and Cdh1 and for APC/C function in vivo. Mutation of these sites increased the rate of activator dissociation from the APC/C but did not affect reaction processivity, suggesting that the mutations have little effect on substrate dissociation from the active site. Further studies revealed that activator dissociation from the APC/C is inhibited by substrate, and that substrates are not bound solely to activator during catalysis but interact bivalently with an additional binding site on the APC/C core.
Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA.
Mary E
ME
Matyskiela
David O
DO
Morgan
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D064196:Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome; D001665:Binding Sites; D064199:Cdc20 Proteins; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000378:metabolism*
D064196:Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome; C577495:CDC20 protein, S cerevisiae; C577441:CDC23 protein, S cerevisiae; C577583:CDH1 protein, S cerevisiae; D064199:Cdc20 Proteins; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome