The class of Cullin–RING E3 ligases (CRLs) selectively ubiquitinate a large portion of proteins targeted for proteolysis by the 26S proteasome. Before degradation, ubiquitin molecules are removed from their conjugated proteins by deubiquitinating enzymes, a handful of which are associated with the proteasome. The CRL activity is triggered by modification of the Cullin subunit with the ubiquitin-like protein, NEDD8 (also known as Rub1 in Saccharomyces cerevisiae). Cullin modification is then reversed by hydrolytic action of the COP9 signalosome (CSN). As the NEDD8–Rub1 catalytic cycle is not essential for the viability of S. cerevisiae, this organism is a useful model system to study the alteration of Rub1–CRL conjugation patterns. In this study, we describe two distinct mutants of Rpn11, a proteasome-associated deubiquitinating enzyme, both of which exhibit a biochemical phenotype characterized by high accumulation of Rub1-modified Cdc53–Cullin1 (yCul1) upon entry into quiescence in S. cerevisiae. Further characterization revealed proteasome 19S-lid-associated deubiquitination activity that authorizes the hydrolysis of Rub1 from yCul1 by the CSN complex. Thus, our results suggest a negative feedback mechanism via proteasome capacity on upstream ubiquitinating enzymes.
Bibliographical noteFunding Information:
Funding: This research was funded by the Israel Ministry of Science and Technology (MOST)–Italian Ministry of Foreign Affairs (MAE) grant 3-9022 to E.P. and T.R.; Israel Science Foundation grants 162/17 for E.P. and 755/19 for M.H.G., and BSF-NSF 2017727 for MHG. L.B. and A.S.’s fellowships and studies at the Pick lab are supported by the Israeli Council for higher education (VATAT).
The CSN5i-3 inhibitor  was provided by Novartis (Novartis Institutes for BioMedical Research, Basel, Switzerland) for Medical Research under the terms of the Novartis Transfer Agreement for academic research proposes. Cultures were grown overnight and diluted in YPD to 0.5 OD600 and grown for 6 h before the addition of CSN5i-3 from a 20 mM stock diluted in DMSO.
© 2019 by the authors. Licensee MDPI, Basel, Switzerland.
- 26S proteasome
- Budding yeast
- CSN (COP9 signalosome)
- Diauxic shift
- NEDD8 (neural precursor cell expressed developmentally down-regulated 8)
- Proteasome lid
- Rub1 (Related ubiquitin 1)
- SCF (Skp, Cullin, F-box containing complex)
- Saccharomyces cerevisiae
ASJC Scopus subject areas
- Molecular Biology