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

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 .

Geminiviruses Subvert Ubiquitination by Altering CSN-Mediated Derubylation of SCF E3 Ligase Complexes and Inhibit Jasmonate Signaling inArabidopsis thaliana

“…It is an appealing hypothesis, however, that the virus might be not only impairing the function of the SCF complexes, but rather also redirecting them toward certain target proteins whose degradation would be advantageous for the viral infection. The fact that the overexpression of a given F-box protein can circumvent the general malfunction of the SCF complexes (Denti et al, 2006;Stuttmann et al, 2009) raises the idea that geminiviruses could be co-opting the SCF-mediated ubiquitination pathway for their own advantage through the promotion of the expression of selected adaptor subunits. It would be interesting to look for F-box proteins upregulated during geminivirus infection or in heterologous expression of geminiviral proteins to localize possible targets of this theoretical mechanism.…”

“…Another possibility would be that the effect of C2 on the SCF complexes could be specific rather than generalized, as suggested by the transcriptomic data. Curiously, Stuttmann et al (2009) propose that defects in cullin derubylation may be tolerated without causing obvious physiological defects.…”

“…Alternatively, a tantalizing, nonexclusive hypothesis could be that C2-mediated blocking of the derubylation of CUL1 could increase the number of CSN complexes available to remove RUB from other cullins, thus reducing CUL3 autodegradation. It has been previously proposed that CSN5A reduced activity could trigger the autoubiquitination and degradation of rubylated CUL1 (Stuttmann et al, 2009). …”

Auxin-Binding Protein 1 is a negative regulator of the SCFTIR1/AFB pathway

“…RNA was extracted from roots of 4-day-old seedlings prior exposure to ethanol vapours and after exposure to ethanol vapours for 8, 24 and 48 h, using a Qiagen RNeasy kit and digested with RNAse-free DNAse on the column following the manufacturer's instructions (Qiagen S.A., Courtaboeuf, France). First-strand complementary DNAs were synthesized from 5 mg of total RNA using oligo-(dT) 20 and SuperScript III reverse transcriptase according to the Protein extraction and western blot analysis. Total proteins were extracted from roots of 4 day post-germination seedlings.…”

“…No difference was observed for TIR1 and AFB1, and only a very subtle decrease of AFB2 and AFB3 mRNA was observed over time. Using immunodetection of the TIR1-HA reporter 20 , no significant change in TIR1 protein was observed in response to ABP1 inactivation, suggesting that ABP1 does not act through quantitative changes of TIR1 (Fig. 2b).…”