Functional Analysis of the RING-Type Ubiquitin Ligase Family of Arabidopsis   

Abstract

Approximately 5% of the Arabidopsis (Arabidopsis thaliana) proteome is predicted to be involved in the ubiquitination/26S proteasome pathway. The majority of these predicted proteins have identity to conserved domains found in E3 ligases, of which there are multiple types. The RING-type E3 is characterized by the presence of a cysteine-rich domain that coordinates two zinc atoms. Database searches followed by extensive manual curation identified 469 predicted Arabidopsis RING domaincontaining proteins. In addition to the two canonical RING types (C3H2C3 or C3HC4), additional types of modified RING domains, named RING-v, RING-D, RING-S/T, RING-G, and RING-C2, were identified. The modified RINGs differ in either the spacing between metal ligands or have substitutions at one or more of the metal ligand positions. The majority of the canonical and modified RING domain-containing proteins analyzed were active in in vitro ubiquitination assays, catalyzing polyubiquitination with the E2 AtUBC8. To help identity regions of the proteins that may interact with substrates, domain analyses of the amino acids outside the RING domain classified RING proteins into 30 different groups. Several characterized protein-protein interaction domains were identified, as well as additional conserved domains not described previously. The two largest classes of RING proteins contain either no identifiable domain or a transmembrane domain. The presence of such a large and diverse number of RING domain-containing proteins that function as ubiquitin E3 ligases suggests that targetspecific proteolysis by these E3 ligases is a complex and important part of cellular regulation in Arabidopsis.The covalent attachment of the 76-amino acid protein ubiquitin to target proteins is a central and essential part of numerous cellular processes in eukaryotes. Ubiquitin attachment is a multistep reaction involving three enzymes referred to as E1, E2, and E3 (Glickman and Ciechanover, 2002). The conjugation cascade begins with the formation of thioester-linked ubiquitin by the ubiquitin-activating enzyme or E1 in an ATPdependent reaction. Thioester-linked ubiquitin is then transferred to a cysteinyl residue of an ubiquitinconjugating (UBC) enzyme or E2. Ubiquitin is then transferred from the E2-ubiquitin intermediate to a lysyl group on the substrate. This final step is mediated by the ubiquitin ligase or E3 enzyme. Uniquely, E3 ligases of the HECT domain class form a ubiquitin thioester prior to transfer to the substrate, whereas all the other ligases interact noncovalently with the E2 carrying a thioester-linked ubiquitin. The E3 is also responsible for recruiting the target protein for ubiquitination, and hence is considered the major substrate recognition component of the pathway.Ubiquitin-dependent protein degradation selectively targets a diverse range of substrates, including receptors, nuclear transcription activators and repressors, abnormal proteins, and other short-lived regulatory proteins for degradation by the 26S proteasome (Glickman and Ciec...

Arabidopsis ubiquitin ligase MIEL1 mediates degradation of the transcription factor MYB30 weakening plant defence

“…Moreover, the total number of N. benthamiana and Arabidopsis nuclei expressing MYB30-CFP is significantly reduced in the presence of MIEL1-YFPv, which rendered FRET-FLIM experiments a challenging task. Functional characterization of MIEL1 showed that this protein is an active E3 ligase able to ubiquitinate MYB30 and that, as previously described for other RING proteins, this activity depends on the integrity of key amino acids in the RING domain, which are essential for E2-dependent protein ubiqutination 8 . Finally, all these findings strongly suggest that epitope-tagging of the proteins does not affect their function.…”

“…2, lane 3). Finally, an previously described active RING-type E3 ligase protein (At5g37270) 8 was not able to ubiquitinate MYB30 in vitro, further demonstrating the specificity of MIEL1-mediated ubiquitination of MYB30 ( Supplementary Fig. S4).…”

“…Here we describe the identification of a cDNA clone encoding a non-characterized Zinc Finger RING-type protein (At5g18650) that we named MIEL1. RING domains are characterized by the presence of a cysteine-rich domain in which, similar to the zinc finger domain, Cys and/or His residues coordinate two zinc ions 8 . RING proteins are classified based on the nature of the metal ligand residues present in the RING domain and/or the number of amino acids between them.…”

“…HECT proteins are unique because they form an Ub-E3 intermediate before transfer of Ub to the substrate 7 . RING and U-Box proteins are structurally related single polypeptides that respectively use zinc chelation and hydrogen bonds/salt bridges to transfer Ub from the Ub-E2 intermediate to the target 8,9 . E3 ligases containing a RING domain can act independently or as part of a multisubunit cullin-RING ligase complex such as SCF (Skp1, Cullin, F-box-type ligase).…”

Ubiquitination-mediated protein degradation and modification: an emerging theme in plant-microbe interactions

“…Like SCF E3s, the RING-type E3 ligases serve as scaffolds to bring together the activated ubiquitin-E2 and the substrate in promoting the transfer of ubiquitin to the substrate. In contrast to that only few HECT-type E3s are identified in plants [32], the SCF and the RING-type E3 are found in hundreds of proteins [33,34]. The U-box is a novel E3 ubiquitin ligase activity-related protein domain that was first identified in the yeast ubiquitination factor UFD2 [35].…”