The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor

Programmed proteolysis of proteins such as mitotic cyclins and Cut2/Pds1p requires a 9-residue conserved motif known as the destruction box (D-box), Strong expression of protein fragments containing destruction boxes, such as the first 70 residues of Cdc13 (N70), inhibits the growth of Schizosaccharomyces pombe at metaphase, This inhibition can be overcome either by removal of all lysine residues from N70 using site-directed mutagenesis (K0-N70) or by raising the concentration of intracellular ubiquitin, Consistent with the idea that competition for ubiquitin accounts for some of its inhibitory effects, wild-type N70 not only stabilized D-box proteins, but also Rum1 and Cdc18, which are degraded by a different pathway, The K0-N70 construct was neither polyubiquitinated nor degraded in vitro, but it blocked the growth of strains of yeast in which anaphase-promoting complex/cyclosome (APC/C) function was compromised by mutation, and specifically inhibited proteolysis of APC/C substrates irt vivo, Both K0-N70 and 20-residue D-box peptides blocked polyubiquitination of other D-box-containing substrates in a cell-free ubiquitination assay system, These data suggest the existence of a D-box receptor protein that recognizes D-boxes prior to ubiquitination.