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

Julia Mergner,Bernhard Kuster,Claus Schwechheimer

doi:10.1074/jbc.m116.767103

Julia Mergner, Bernhard Kuster + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m116.767103

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Abstract

In eukaryotes, the conjugation of the ubiquitin-like protein NEDD8 onto protein targets is an important post-translational modification. The best understood neddylation targets are the cullins, scaffold subunits of E3 ubiquitin ligases, where neddylation as well as deneddylation, facilitated by the protease activity of the CSN (COP9 signalosome), are required to control ubiquitin ligase assembly, function, and ultimately substrate degradation. Little is known about the role of other deneddylating enzymes besides CSN and the role of neddylation and deneddylation of their substrates. We previously characterized Arabidopsis thaliana mutants with defects in the conserved NEDD8-specific protease DEN1 (DENEDDYLASE1). These mutants display only subtle growth phenotypes despite the strong accumulation of a broad range of neddylated proteins. Specifically, we identified AXR1 (AUXIN-RESISTANT1), a subunit of the heterodimeric NAE (E1 NEDD8-ACTIVATING ENZYME), as highly neddylated in den1 mutants. Here, we examined the mechanism and consequences of AXR1 neddylation in more detail. We find that AXR1 as well as other neddylation enzymes are autoneddylated at multiple lysines. NAE autoneddylation can be linked to reduced NCE (E2 NEDD8-CONJUGATING ENZYME) NEDD8 thioester levels, either by critically reducing the pool of free NEDD8 or by reducing NAE activity. In planta, increasing NEDD8 gene dosage is sufficient to suppress den1 mutant phenotypes. We therefore suggest that DEN1 serves to recover diverted NEDD8 moieties from autoneddylated NAE subunits, and possibly also other neddylated proteins, to maintain NEDD8 pathway activity toward other NEDD8-dependent processes such as cullin E3 ligase regulation.

Highlights

In eukaryotes, the conjugation of the ubiquitin-like protein NEDD8 onto protein targets is an important post-translational modification
We have previously reported that Arabidopsis AXR1 can be neddylated in the wild type and that AXR1 neddylation is strongly increased in the den1 mutant
Our analyses revealed that the NEDD8 E1-activating enzymes (NAE) subunit ECR1 as well as the NCE enzyme RCE1 can be NEDD8-modified in a manner similar to AXR1 in vitro, with a different efficiency in vivo

Summary

Edited by Joseph Jez

The conjugation of the ubiquitin-like protein NEDD8 onto protein targets is an important post-translational modification. We previously characterized Arabidopsis thaliana mutants with defects in the conserved NEDD8-specific protease DEN1 (DENEDDYLASE1) These mutants display only subtle growth phenotypes despite the strong accumulation of a broad range of neddylated proteins. We identified AXR1 (AUXIN-RESISTANT1), a subunit of the heterodimeric NAE (E1 NEDD8-ACTIVATING ENZYME), as highly neddylated in den mutants. We suggest that DEN1 serves to recover diverted NEDD8 moieties from autoneddylated NAE subunits, and possibly other neddylated proteins, to maintain NEDD8 pathway activity toward other NEDD8-dependent processes such as cullin E3 ligase regulation. NEDD8 is covalently attached to its substrate through an isopeptide bond involving the C-terminal Gly (glycine) of NEDD8 and an ε-amino group of a Lys (lysine) in the target protein [8] Cleavage of this bond and removal of the modification is achieved through the activities of specific deubiquitinating iso-peptidases, so-called DUBs [10]. Our data indicate that DEN1 functions to maintain NEDD8 pathway activity by recovering diverted NEDD8 moieties from proteins of the NEDD8 conjugation machinery

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Experimental Procedures