DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

Abstract

The evolutionarily conserved 8-kD protein NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8) belongs to the family of ubiquitin-like modifiers. Like ubiquitin, NEDD8 is conjugated to and deconjugated from target proteins. Many targets and functions of ubiquitylation have been described; by contrast, few targets of NEDD8 have been identified. In plants as well as in non-plant organisms, the cullin subunits of cullin-RING E3 ligases are NEDD8 conjugates with a demonstrated functional role for the NEDD8 modification. The existence of other non-cullin NEDD8 targets has generally been questioned. NEDD8 is translated as a precursor protein and proteolytic processing exposes a C-terminal glycine required for NEDD8 conjugation. In animals and yeast, DENEDDYLASE1 (DEN1) processes NEDD8. Here, we show that mutants of a DEN1 homolog from Arabidopsis thaliana have no detectable defects in NEDD8 processing but do accumulate a broad range of NEDD8 conjugates; this provides direct evidence for the existence of non-cullin NEDD8 conjugates. We further identify AUXIN RESISTANT1 (AXR1), a subunit of the heterodimeric NEDD8 E1 activating enzyme, as a NEDD8-modified protein in den1 mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins.

DENEDDYLASE1 Protein Counters Automodification of Neddylating Enzymes to Maintain NEDD8 Protein Homeostasis in Arabidopsis

“…Our previous comparative analyses of den1 and csn5 mutants from Arabidopsis thaliana showed that, at least in Arabidopsis, DEN1 is specifically required to remove NEDD8 from neddylated substrates other than cullins (31). We further identified AXR1 and, indirectly, also the paralogous AXL as prominent NEDD8-modified proteins that depend on DEN1 for deneddylation (31).…”

“…We further identified AXR1 and, indirectly, also the paralogous AXL as prominent NEDD8-modified proteins that depend on DEN1 for deneddylation (31). AXR1 and AXL share ϳ80% amino acid identity, and both can form a complex with ECR1 (E1 C-TERMINAL RELATED1) (23,32).…”

“…DEN1 was originally isolated as a NEDD8-specific C-terminal processing enzyme based on its in vitro activity (26). However, NEDD8 processing is not detectably impaired in den1 mutants from fission yeast (Schizosaccharomyces pombe), fungi (Aspergillus nidulans), Arabidopsis, Drosophila, or mice, suggesting that the in vivo activity of DEN1 may be different from its reported in vitro activity, or that other functionally redundant proteases can process NEDD8 in den1 mutants (27)(28)(29)(30)(31). Because den1 mutants from different species accumulate high levels of neddylated substrates of a broad molecular range, it was instead concluded that DEN1 serves in vivo to deconjugate NEDD8 from neddylated substrates (27)(28)(29)(30)(31).…”

“…Adult axr1 plants are dwarfed, have reduced apical dominance, curled leaves, and reduced fertility (11,33,34). While den1 mutants show no obvious growth defects, the den1 mutation has a clear effect on plant growth in an AXR1-sensitized background, specifically, an axr1-30 Myc:AXR1 complementation line where the expression of a Myc-tagged AXR1 transgene only partially rescues the axr1-30 mutant phenotype (31,32). This suggests that AXR1 neddylation negatively affects the neddylation pathway in den1 mutant backgrounds.…”

It’s Time for Some “Site”-Seeing: Novel Tools to Monitor the Ubiquitin Landscape in Arabidopsis thaliana

“…For instance, because USP2cc recognizes the last five amino acids of ubiquitin and no other ubiquitin-like proteins harbor this sequence at their C termini, this DUB is ubiquitin specific in plants, in contrast to the K-e-GG antibodies that also recognize the epitope generated upon tryptic cleavage of other small protein modifiers, such as RELATED TO UBIQUITIN (Vierstra, 2012). Currently, it is difficult to assess to which extent this ambiguity is problematic because the degree of rubbylation in plants is unknown, but its functionality has been suggested to supersede that of neddylation, its mammalian PTM counterpart (Hakenjos et al, 2011;Mergner and Schwechheimer, 2014;Mergner et al, 2015).…”