The NEDD8 modification pathway in plants

Abstract

NEDD8, in plants and yeasts also known as RELATED TO UBIQUITIN (RUB), is an evolutionarily conserved 76 amino acid protein highly related to ubiquitin. Like ubiquitin, NEDD8 can be conjugated to and deconjugated from target proteins, but unlike ubiquitin, NEDD8 has not been reported to form chains similar to the different polymeric ubiquitin chains that have a role in a diverse set of cellular processes. NEDD8-modification is best known as a post-translational modification of the cullin subunits of cullin-RING E3 ubiquitin ligases. In this context, structural analyses have revealed that neddylation induces a conformation change of the cullin that brings the ubiquitylation substrates into proximity of the interacting E2 conjugating enzyme. In turn, NEDD8 deconjugation destabilizes the cullin RING ligase complex allowing for the exchange of substrate recognition subunits via the exchange factor CAND1. In plants, components of the neddylation and deneddylation pathway were identified based on mutants with defects in auxin and light responses and the characterization of these mutants has been instrumental for the elucidation of the neddylation pathway. More recently, there has been evidence from animal and plant systems that NEDD8 conjugation may also regulate the behavior or fate of non-cullin substrates in a number of ways. Here, the current knowledge on NEDD8 processing, conjugation and deconjugation is presented, where applicable, in the context of specific signaling pathways from plants.

Keywords: CAND1; COP9 signalosome (CSN); E3 ubiquitin ligase; F-BOX PROTEIN (FBP); NEDD8; RELATED TO UBIQUITIN (RUB); cullin; ubiquitin.

FIGURE 1

Protein domain organization of NEDD8 proteins from several representative species. Gene identification numbers (Gene IDs) are as listed in www.ensemblgenomes.org. Specifically indicated are the last five amino acids of the respective proteins before the proteolytic cleave sites, the first amino acid of NEDD8 and the proteins’ C-terminal amino acids. Proteolytic processing occurs after the C-terminal RGG residues and is indicated by an underscore. Positions of introns in the respective genes are indicated by a triangle. The light gray area in BRADI4G28550 highlights an apparent 22 amino acid deletion in the ubiquitin part of the protein. A. thaliana (Arabidopsis thaliana), O. sativa (Oryza sativa, rice), B. distachyon (Brachypodium distachyon), P. patens (Physcomitrella patens, moss), C. reinhardtii (Chlamydomonas reinhardtii, algae), D. melanogaster (Drosophila melanogaster, fruit fly), S. cerevisiae (Saccharomyces cerevisiae, baker’s yeast), S. pombe (Schizosaccharomyces pombe, fission yeast).

FIGURE 2

Neddylation and ubiquitin modification are biochemically related processes. Ubiquitin c-terminal hydrolases (UCHs) belong to the family of DUBs that process ubiquitin (UBI)-NEDD8 fusion proteins. UBI and NEDD8 are activated by their conjugation to E1 ubiquitin/NAEs. UBI or NEDD8 from the E1 are then passed via a transthiolation reaction to a protein of the family of E2 ubiquitin-conjugating enzymes, RUB1 CONJUGATING ENZYME1 (RCE1) in Arabidopsis. The ubiquitin-charged E2 can then form a complex with an E3 ubiquitin ligase, and ultimately, ubiquitin and NEDD8 are transferred to a lysine residue on the cullin of the E3 and the substrate, respectively. UBI but not NEDD8 can form chains. RBX1 and other proteins not shown here are the proposed NEDD8 ligases. Cullin-RING ligases (CRLs) are the superfamily of E3 ubiquitin ligases that are regulated by neddylation. CRLs are generally composed of a cullin subunit, RBX1, as well as a substrate (SUB) recognition module composed of an adaptor and a substrate receptor protein. C, cysteine; K, lysine.

FIGURE 3

NEDD8 processing is mediated by at least three different classes of peptidases. Unrooted phylogenetic tree of representative members of the UCH and DEN1/NEDP1/SENP8 protein families from Arabidopsis thaliana (At), rice (Oryza sativa, Os), algae (Chlamydomonas reinhardtii, Cr), baker’s yeast (Saccharomyces cerevisiae, Sc), fission yeast (Schizosaccharomyces pombe, Sp), fruit fly (Drosophila melanogaster, Dm), mouse (Mus musculus, Mm), and human (Homo sapiens, Hs). Molecular phylogenetic analysis was performed based on the Maximum Likelihood method and the JTT matrix-based model (Jones et al., 1992) using CLUSTALW algorithm at the EMBL-EBI website (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The tree with the highest log likelihood (-5916.8263) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying the Maximum Parsimony method. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 24 protein sequences. All positions containing gaps and missing data were eliminated. There were a total of 145 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).

FIGURE 4

CAND1 regulates the cells CRL repertoire by promoting the exchange of substrate receptor subunits. Schematic representation of the exchange of a hypothetical F-BOX PROTEIN1 (FBP1) of the CRL SCF^FBP1 against FBP2 following CULLIN1 deneddylation. ARABIDOPSIS SKP1 (ASK) proteins are adaptor subunits that link FBPs with CULLIN1. CULLIN1, ASK, RBX1 and FBP form an SCF-type CRL. COP9 SIGNALOSOME (CSN) promotes CULLIN deneddylation. CULLIN-ASSOCIATED-NEDD8-DISSOCIATED1 (CAND1) is an exchange factor that can only associate with deneddylated cullins and promotes substrate receptor exchange. C, cysteine; K, lysine.