Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC.
Ubiquitin-mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi-subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate-specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co-immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo-related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C-box, a motif required for APC association of Hct1 and conserved among Cdc20-related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.
Schwab
M
M
Institute of Industrial Genetics, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Neutzner
M
M
Möcker
D
D
Seufert
W
W
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D064199:Cdc20 Proteins; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D019926:Cyclin B / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008025:Ligases / Q000378:metabolism*; D008938:Mitosis; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D064249:Securin; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
C577495:CDC20 protein, S cerevisiae; C577583:CDH1 protein, S cerevisiae; C093749:CLB2 protein, S cerevisiae; D064199:Cdc20 Proteins; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins; D019926:Cyclin B; D005656:Fungal Proteins; D009687:Nuclear Proteins; C578004:PDS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D064249:Securin; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C081927:CDC5 protein, S cerevisiae; D008025:Ligases