The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase. The COP9 signalosome (CSN) complex was originally identified as a negative regulator of photomorphogenesis in plants (for review, see Schwechheimer and Deng 2001). Subsequently, it was purified from human cell extracts during attempts to isolate the 19S regulatory lid complex of the proteosome (Seeger et al. 1998). The human signalosome consists of eight core subunits, each sharing significant homology with a corresponding subunit in the regulatory 19S lid complex of the proteosome . The purified CSN complex can cleave the ubiquitin-like Nedd8 protein from cullins Wee et al. 2002). Csn5 contains a putative metalloprotease motif that is presumed to mediate deneddylation activity (Cope et al. 2002). Cullins are subunits of E3 ubiquitin ligases (Feldman et al. 1997;Skowyra et al. 1997), and deneddylation of cullins decreases SCF E3 ubiquitin ligase activity (Osaka et al. 2000). SCF E3 complexes typically consist of a cullin, the Rbx1 RING domain protein that binds an E2 enzyme Skowyra et al. 1999), and an adapter protein, Skp1, that binds an F-box protein that determines the substrate specificity (Skowyra et al. 1997).In Arabidopsis, the signalosome is involved in the degradation of the two bZIP transcription factors (Hy5, HyH) that lie at the top of a transcriptional cascade required to induce ∼ 30% of Arabidopsis genes during photomorphogenesis (Holm et al. 2002). An E2-like protein, Cop10, and an E3 RING protein, Cop1, are also required to degrade Hy5 and HyH, which occurs when seedlings are germinated in the dark (Osterlund et al. 2000;Holm et al. 2002;Suzuki et al. 2002). The biochemical role of the signalosome is unknown, although a correlation with Cop1 nuclear localization (von Arnim et al. 1997) and the associations between the signalosome and E3 ubiquitin ligases Schwechheimer et al. 2002) suggest a regulatory role in ubiquitination that may be linked to subcellular localization (Chamovitz et al. 1996;Hellmann and Estelle 2002).A highly conserved signalosome complex was identified in the fission yeast Schizosaccharomyces pombe (Mundt et al. 1999) and subsequently shown to be required to remove the Nedd8 ubiquitin-like protein fr...