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 El-like enzyme (APP-BP1/Uba3), NEDD8. specific E2 enzyme (Ubc 12) 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 oil 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 Ubc 12 to Cul-1. These results Suggest the important regulatory role of ROC1 for poly-NEDD8 chain formation, (C) 2009 Elsevier Inc. All rights reserved.