COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation in Arabidopsis

Pascal Genschik,Johannes Stuttmann,Laurent D. Noël,Esther Lechner,Laurent Nussaume,Jane E. Parker,Raphael Guérois

doi:10.1074/jbc.m809069200

Abstract

Ubiquitination and proteasome-mediated degradation of proteins are crucial for eukaryotic physiology and development. The largest class of E3 ubiquitin ligases is made up of the cullin-RING ligases (CRLs), which themselves are positively regulated through conjugation of the ubiquitin-like peptide RUB/NEDD8 to cullins. RUB modification is antagonized by the COP9 signalosome (CSN), an evolutionarily conserved eight-subunit complex that is essential in most eukaryotes and cleaves RUB from cullins. The CSN behaves genetically as an activator of CRLs, although it abolishes CRL activity in vitro. This apparent paradox was recently reconciled in different organisms, as the CSN was shown to prevent autocatalytic degradation of several CRL substrate adaptors. We tested for such a mechanism in the model plant Arabidopsis by measuring the impact of a newly identified viable csn2 mutant on the activity and stability of SCF(TIR1), a receptor to the phytohormone auxin and probably the best characterized plant CRL. Our analysis reveals that not only the F-box protein TIR1 but also relevant cullins are destabilized in csn2 and other Arabidopsis csn mutants. These results provide an explanation for the auxin resistance of csn mutants. We further observed in vivo a post-translational modification of TIR1 dependent on the proteasome inhibitor MG-132 and provide evidence for proteasome-mediated degradation of TIR1, CUL1, and ASK1 (Arabidopsis SKP1 homolog). These results are consistent with CSN-dependent protection of Arabidopsis CRLs from autocatalytic degradation, as observed in other eukaryotes, and provide evidence for antagonist roles of the CSN and 26S proteasome in modulating accumulation of the plant CRL SCF(TIR1).

Highlights

SGT1 is needed for optimal activity of several SCFs including SCFTIR1 in Arabidopsis [41], its exact mode of action on SCFs remains unclear. 3100 individual M2 families originating from ethane methyl sulfonate-mutagenized Landsberg erecta (Ler) sgt1b-3 mutant seeds were first germinated on auxin-free medium and screened for enhanced auxin resistance in a root growth inhibition assay using 0.2 ␮M synthetic auxin 2,4-D
We investigated the post-translational regulation of CRL ubiquitin ligases in both wild-type plants and csn mutants, focusing on the auxin receptor SCFTIR1
We show that the COP9 signalosome (CSN) is needed for the stabilization of cullins and other CRL subunits in Arabidopsis and that SCF components, including the substrate adaptor TIR1, are degraded by the ubiquitin/proteasome machinery even in wild-type plants

Summary

Introduction

To test the dependence of the csn2-5 auxin resistance on the sgt1b-3 background, the double mutant was out-crossed to Ler wild-type plants and the csn2-5 single mutant selected. The csn5a-1 mutant, which carries a null mutation in one of two genes encoding the MPN subunit 5 of the CSN complex, is a severe dwarf [40] and displays similar defects in auxin signaling, cullin accumulation, and RUB modification as the csn2-5 mutant (supplemental Fig. 2, A and B).

Results

Conclusion