The mechanism of poly-NEDD8 chain formation in vitro.
NEDD8 is a small ubiquitin-like protein that modifies target proteins in a reaction similar to ubiquitination. In this reaction, three enzymes are required and sufficient: NEDD8 activating E1-like enzyme (APP-BP1/Uba3), NEDD8-specific E2 enzyme (Ubc12) and RING-finger protein ROC1 (NEDD8 E3 ligase). Unlike ubiquitin, which is well known to form poly-ubiquitin chain, little is known about the formation of poly-NEDD8 chain. Here we show the mechanism of poly-NEDD8 chain formation on Cullin-1 using a complete in vitro reconstituted NEDD8 conjugation system. Intriguingly, poly-NEDD8 chain was build up on catalytic Cysteine residue of Ubc12. Furthermore, Ubc12 formed poly-NEDD8 chain without the activity of ROC1. Rather ROC1 mutant, defective for ubiquitin ligase activity, dramatically enhanced the poly-NEDD8 chain formation. In turn, ROC1 was essential for the transfer of poly-NEDD8 chain from Ubc12 to Cul-1. These results suggest the important regulatory role of ROC1 for poly-NEDD8 chain formation.
Ohki
Yu
Y
Department of Molecular Biology, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan.
Funatsu
Norikazu
N
Konishi
Natsumi
N
Chiba
Tomoki
T
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D044842:Cullin Proteins / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D000075687:NEDD8 Protein; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D002352:Carrier Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D011108:Polymers; C479529:RBX1 protein, human; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D003545:Cysteine