1. Institute of Biochemistry, ETH Hönggerberg, 8093 Zürich, Switzerland
2. Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA
3. Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
4. Memorec Biotec GmbH, 50829 Köln, Germany

Correspondence to: Bruce Bowerman²Matthias Peter¹ Correspondence and requests for materials should be addressed to B.B. (Email: bbowerman@molbio.uoregon.edu) or M.P. (Email: matthias.peter@bc.biol.ethz.ch).

Abstract

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome¹. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module^{1, 2, 3, 4}. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation)⁵ and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity^{6, 7}. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.

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