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...

Using electroporation-mediated gene transfer, the gene encoding the Slow (S) migrating polypeptide of the maize (Zea mays L.) alcohol dehydrogenase-1 (Adhl) enzyme has been introduced stably and transiently into maize cells containing an endogenous Fast (F) ADH1 electromorph. In stable transformants an l l.5-kb fragment was sufficient to program normal S expression relative to the endogenous F allele. In transient assays, Adhl-S gene constructs lacking the 9 Adhl-S intervening sequences (introns) were expressed at levels 50-to 100-fold less than the intact gene; the presence of intron 1 alone restored levels of gene expression to those found with the intact gene. The last two introns also stimulate Adhl-S expression, but the level is threefold below that of the intact gene. The expression of a chimeric chloramphenicol acetyltransferase (CAT) gene utilizing the 5' promoter and 3' polyadenylation regions of the Adhl gene was increased 100-fold by the addition of sequences containing the Adhl intron 1. The Adhl intron 1 sequences did not stimulate CAT expression when located outside the transcribed region. When located within the transcribed region, the Adhl intron 1 region efficiently stimulated CAT expression only when located between the promoter and the CAT coding region. A construct containing the Adhl intron 1 fragment produced 40-fold more mRNA than a construct containing an equivalent cDNA fragment. Both the Adhl intron 1 and the intron from a second maize gene, Bronzel, stimulated expression from other promoters (cauliflower mosaic virus 355 and nopaline synthase) and of other coding regions (luciferase and neomycin phosphotransferase II) as well. These results indicated that introns increase both Adhl and chimeric gene expression in maize and the optimal location for such an intron is near the 5' end of the mRNA.

Analysis of the Arabidopsis thaliana RING-ANK (for Really Interesting New Gene-Ankyrin) family, a subgroup of RING-type E3 ligases, identified KEEP ON GOING (KEG) as essential for growth and development. In addition to the RING-HCa and ankyrin repeats, KEG contains a kinase domain and 12 HERC2-like repeats. The RING-HCa and kinase domains were functional in in vitro ubiquitylation and phosphorylation assays, respectively. Seedlings homozygous for T-DNA insertions in KEG undergo growth arrest immediately after germination, suggestive of increased abscisic acid (ABA) signaling, a major phytohormone that plays a key role in plant development and survival under unfavorable conditions. Here, we show that KEG is a negative regulator of ABA signaling. keg roots are extremely sensitive to the inhibitory effects of ABA and exhibit hypersensitivity to exogenous glucose, consistent with the known interaction between glucose and ABA signaling. The observations that KEG accumulates high levels of ABSCISIC ACID-INSENSITIVE5 (ABI5) without exogenous ABA, interacts with ABI5 in vitro, and that loss of ABI5 rescues the growth-arrest phenotype of keg mutant seedlings indicate that KEG is required for ABI5 degradation. In this capacity, KEG is central to ABA signaling by maintaining low levels of ABI5 in the absence of stress.

The COP9 signalosome is an evolutionary conserved multiprotein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. Here, we show that plants with reduced COP9 signalosome levels had decreased auxin response similar to loss-of-function mutants of the E3 ubiquitin ligase SCFTIR1. Furthermore, we found that the COP9 signalosome and SCFTIR1 interacted in vivo and that the COP9 signalosome was required for efficient degradation of PSIAA6, a candidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses.

Epigenetic gene silencing suppresses transposon activity and is critical for normal development . Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2). In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif.