Assembly of an APC-Cdh1-substrate complex is stimulated by engagement of a destruction box.
The anaphase-promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators. Cdh1p is an APC coactivator that directly binds APC substrates. A genetic screen in budding yeast identified residues within Cdh1p critical for its function. Cdh1p proteins containing mutations within the "C box" or the "IR" motif could bind substrate, but not the APC, whereas mutants that only bound the APC were not identified, suggesting an ordered assembly of the ternary APC-Cdh1p-substrate complex. Supporting this hypothesis, we found that substrate binding to wild-type Cdh1p enhanced its association with the APC in yeast cells. We used peptide competition assays to demonstrate that Cdh1p interacts directly with the D box and the KEN box, two motifs within APC substrates known to be required for APC-mediated degradation. Moreover, an intact D box domain within a substrate was required to stimulate the association between the Cdh1p-substrate complex and the APC.
Burton
Janet L
JL
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Tsakraklides
Vasiliki
V
Solomon
Mark J
MJ
D020816:Amino Acid Motifs; D064173:Anaphase-Promoting Complex-Cyclosome; D064200:Cdh1 Proteins; D004252:DNA Mutational Analysis; D046912:Multiprotein Complexes; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics* / Q000378:metabolism*
C577583:CDH1 protein, S cerevisiae; D064200:Cdh1 Proteins; D046912:Multiprotein Complexes; D010455:Peptides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D011494:Protein Kinases; C106494:HSL1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases