Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans.
BACKGROUND
SCF (Skp1-Cullin-F-box) complexes are a major class of E3 ligases that are required to selectively target substrates for ubiquitin-dependent degradation by the 26S proteasome. Conjugation of the ubiquitin-like protein Nedd8 to the cullin subunit (neddylation) positively regulates activity of SCF complexes, most likely by increasing their affinity for the E2 conjugated to ubiquitin. The Nedd8 conjugation pathway is required in C. elegans embryos for the ubiquitin-mediated degradation of the microtubule-severing protein MEI-1/Katanin at the meiosis-to-mitosis transition. Genetic experiments suggest that this pathway controls the activity of a CUL-3-based E3 ligase. Counteracting the Nedd8 pathway, the COP9/signalosome has been shown to promote deneddylation of the cullin subunit. However, little is known about the role of neddylation and deneddylation for E3 ligase activity in vivo.


RESULTS
Here, we identified and characterized the COP9/signalosome in C. elegans and showed that it promotes deneddylation of CUL-3, a critical target of the Nedd8 conjugation pathway. As in other species, the C. elegans signalosome is a macromolecular complex containing at least six subunits that localizes in the nucleus and the cytoplasm. Reducing COP9/signalosome function by RNAi results in a failure to degrade MEI-1, leading to severe defects in microtubule-dependent processes during the first mitotic division. Intriguingly, reducing COP9/signalosome function suppresses a partial defect in the neddylation pathway; this suppression suggests that deneddylation and neddylation antagonize each other.


CONCLUSIONS
We conclude that both neddylation and deneddylation of CUL-3 is required for MEI-1 degradation and propose that cycles of CUL-3 neddylation and deneddylation are necessary for its ligase activity in vivo.
Pintard
Lionel
L
Institute of Biochemistry, HPM G.10.1, ETH Hönggerberg, 8093 Zürich, Switzerland.
Kurz
Thimo
T
Glaser
Sarah
S
Willis
John H
JH
Peter
Matthias
M
Bowerman
Bruce
B
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D000075686:COP9 Signalosome Complex; D017173:Caenorhabditis elegans / Q000378:metabolism / Q000502:physiology; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D044842:Cullin Proteins / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D000074201:Katanin; D008540:Meiosis / Q000502:physiology; D008938:Mitosis / Q000502:physiology; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D010447:Peptide Hydrolases; D011494:Protein Kinases / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D020798:Two-Hybrid System Techniques; D014452:Ubiquitins / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D044842:Cullin Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D011506:Proteins; D014452:Ubiquitins; D011494:Protein Kinases; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex; D000251:Adenosine Triphosphatases; C086678:MEI-1 protein, C elegans; D000074201:Katanin