SCF is a ubiquitin ligase and is composed of Skp1, Cull, F-box protein, and Roc1. The catalytic site of the SCF is the Cull/Roc1 complex and RING-finger protein Roc1. It was shown earlier that when Cull was co-expressed with Roc1 in Sf-9 cells in a baculovirus protein expression system, Cull was highly neddylated in the cell, suggesting that Roc1 may function as a Nedd8-E3 ligase. However, there is no direct evidence that Roc1 is a Nedd8-E3 in an in vitro enzyme system. Here we have shown that Roc1 binds to Ubc12, E2 for Nedd8, but not to Ubc9, E2 for SUMO-1 and Roc1 RING-finger mutant, H77A, did not bind to Ubc12. In in vitro neddylation system using purified Cu11/Roc1 complex expressed in bacteria, Roc1 promotes neddylation of Cull. These results demonstrate that Roc1 functions as a Nedd8-E3 ligase toward Cull. Furthermore, Roc1 and Cu11 were ubiquitinylated in a manner dependent on the neddylation of Cu11 in vitro. In addition, Cu11 was degraded through the ubiquitin-proteasome pathway, and a non-neddylated mutant Cu11, K720R, was more stable than wild-type in intact cells. Thus, neddylation of Cu11 might regulate SCF function negatively via degradation of Cu11/Roc1 complex. (C) 2003 Elsevier Science (USA). All rights reserved.