The Nedd8-conjugated ROC1-CUL1 core ubiquitin ligase utilizes Nedd8 charged surface residues for efficient polyubiquitin chain assembly catalyzed by Cdc34.
Lysine 48-linked polyubiquitin chains are the principle signal for targeting proteins for degradation by the 26 S proteasome. Here we report that the conjugation of Nedd8 to ROC1-CUL1, a subcomplex of the SCF-ROC1 E3 ubiquitin ligase, selectively stimulates Cdc34-catalyzed lysine 48-linked multiubiquitin chain assembly. We have further demonstrated that separate regions within the human Cdc34 C-terminal tail are responsible for multiubiquitin chain assembly and for physical interactions with the Nedd8-conjugated ROC1-CUL1 to assemble extensive ubiquitin polymers. Structural comparisons between Nedd8 and ubiquitin reveal that six charged residues (Lys4, Glu12, Glu14, Arg25, Glu28, and Glu31) are uniquely present on the surface of Nedd8. Replacement of each of the six residues with the corresponding amino acid in ubiquitin decreases the ability of Nedd8 to activate the ubiquitin ligase activity of ROC1-CUL1. Moreover, maintenance of the proper charges at amino acid positions 14 and 25 are necessary for retaining wild type levels of activity, whereas introduction of the opposite charges at these positions abolishes the Nedd8 activation function. These results suggest that Nedd8 charged surface residues mediate the activation of ROC1-CUL1 to specifically support Cdc34-catalyzed ubiquitin polymerization.
Wu
Kenneth
K
Derald H. Ruttenberg Cancer Center, The Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
Chen
Angus
A
Tan
Peilin
P
Pan
Zhen-Qiang
ZQ
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D002384:Catalysis; D008025:Ligases / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D011108:Polymers / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000737:chemistry / Q000502:physiology*
D011108:Polymers; D025801:Ubiquitin; D014452:Ubiquitins; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases