Selective degradation of ubiquitinated Sic1 by purified 26S proteasome yields active S phase cyclin-Cdk.
Selective degradation of single subunits of multimeric complexes by the ubiquitin pathway underlies multiple regulatory switches, including those involving cyclins and Cdk inhibitors. The machinery that segregates ubiquitinated proteins from unmodified partners prior to degradation remains undefined. We report that ubiquitinated Sic1 (Ub-Sic1) embedded within inactive S phase cyclin-Cdk (S-Cdk) complexes was rapidly degraded by purified 26S proteasomes, yielding active S-Cdk. Mutant proteasomes that failed to degrade Ub-Sic1 activated S-Cdk only partially in an ATP-dependent manner. Whereas Ub-Sic1 was degraded within approximately 2 min, spontaneous dissociation of Ub-Sic1 from S-Cdk was approximately 200-fold slower. We propose that the 26S proteasome has the intrinsic capability to extract, unfold, and degrade ubiquitinated proteins while releasing bound partners untouched. Activation of S-Cdk reported herein represents a complete reconstitution of the regulatory switch underlying the G1/S transition in budding yeast.
Verma
R
R
Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
McDonald
H
H
Yates
J R
JR
Deshaies
R J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D016196:S Phase / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D004718:Saccharomycetales / Q000502:physiology; D014452:Ubiquitins / Q000378:metabolism*
D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D021122:Protein Subunits; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D018844:Cyclin-Dependent Kinases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; C080044:PRE2 protein, S cerevisiae; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease