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Plant Physiology 137:13-30 (2005) © 2005 American Society of Plant Biologists Functional Analysis of the RING-Type Ubiquitin Ligase Family of Arabidopsis1,[w]Section of Molecular and Cellular Biology, Division of Biological Sciences (S.L.S., H.H., A.T., J.H., E.K., J.C.), and Plant Biology Graduate Group Program (E.K., J.C.), University of California, Davis, California 95616
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 domain-containing 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 target-specific 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 ATP-dependent reaction. Thioester-linked ubiquitin is then transferred to a cysteinyl residue of an ubiquitin-conjugating (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 Ciechanover, 2002
More than 5% of the predicted Arabidopsis (Arabidopsis thaliana) proteome is postulated to be involved in the ubiquitination/26S proteasome pathway (Smalle and Vierstra, 2004
The Arabidopsis E3 ligases can be grouped into three defined classes based on the presence of the HECT, U-box, or RING domain (Smalle and Vierstra, 2004
The Cys-rich RING domain was first identified in a protein encoded by the Really Interesting New Gene, hence the domain name (Freemont et al., 1991
The Arabidopsis genome is predicted to encode for a considerable number of RING proteins of which very little is known (Kosarev et al., 2002 The large number of potential RING proteins and other types of E3 ligases suggests that target specific ubiquitination plays an important role in protein regulation in Arabidopsis. However, there is little evidence demonstrating that the Arabidopsis family of RING domain-containing proteins functions as E3 ligases. Therefore, in this study, we carried out an extensive search of the Arabidopsis genome to identify potential RING-containing proteins and tested whether representative family members possess E3 ligase activity. In addition to the canonical RING domains, the search also led to the identification of a number of modified RING domains. Here, we also describe the domain organization of each RING and modified RING domain-containing protein. Finally, we provide biochemical evidence that a significant number of the 469 RING and modified RING proteins identified have the capacity for E2-dependent protein ubiquitination and that the RING domain is required for this activity.
Predicted RING Domain-Containing Proteins in Arabidopsis Can Be Divided into Three RING Types and Five Modified RING Types
Our analysis of the translated Arabidopsis genome resulted in a final collection of 477 RING domains in 469 predicted proteins containing one or more of the various types of RING domains (Fig. 1A). A complete list of all RING domain-containing proteins identified can be found in Supplemental Table I. RING domain-containing proteins were identified by database searches via InterPro (release v7.2), which compiles the results from multiple domain-searching programs. A number of additional loci were identified using reiterative BLAST searches of the nonredundant Arabidopsis genome (The Arabidopsis Information Resource [TAIR] database, release date April 16, 2003; Rhee et al., 2003
The 477 RING domains were classified into one of eight RING types based on the nature of the metal ligand residues present and/or the number of amino acids between them (Fig. 1A). The largest was the RING-H2 type with 241 domains, followed by the RING-HC type with 186 domains (Fig. 1A). The RING-H2 class includes AtRBX1a and AtRBX1b, which, as observed in other Rbx/Roc/Hrt proteins, has an Asp residue at metal ligand position 8 instead of a Cys residue and a number of additional Cys residues between metal ligands 2 and 3 that have been shown to form a third zinc ion-binding site (Supplemental Fig. 1; Kamura et al., 1999 ; Chen et al., 2000; Zheng et al., 2002). This variation is unique to the Rbx family of proteins and is not observed in any other RING-H2 protein (data not shown). The RING-HC class was divided into two subgroups: the RING-HCa and RING-HCb, with 145 and 41 domains, respectively (Figs. 1A and 2). All RING-HCb domains contain additional amino acids between metal ligand residues C7 and C8 (Fig. 1A; Supplemental Table I). The division of the RING-HC class is further supported by the difference in the range of amino acids found between metal ligands 2 and 3 and metal ligands 6 and 7 (Fig. 1C). More than 70% of the RING-HCa domains contain 12 amino acids between metal ligands 2 and 3, whereas only approximately 18% of RING-HCb domains have 12 amino acids between these metal ligands. The majority of RING-HCb domains have 14 to 16 amino acids between metal ligands 2 and 3. In addition, the number of amino acids between metal ligands 6 and 7 of the RING-HCb domain is greater than that of the RING-HCa (Fig. 1C). Finally, when the entire amino acid sequences of RING-HCb proteins are compared, these proteins share additional predicted protein-protein interaction domains (Supplemental Table I), again suggesting a closer relationship among them than to RING-HCa proteins. The RING-HCb group includes members of the ARIADNE (ARI) protein family described previously (Mladek et al., 2003).
The modified RING types represent only 11% of the total predicted RING domains identified (Fig. 1A). Previously described modified RING domains found in Arabidopsis include the RING-v, RING-C2, and RING-G types. The RING-v domain (Hewitt et al., 2002 ) is characterized by a cysteinyl residue at metal ligand position 4 and a histinyl residue at metal ligand position 5, rather than the reverse found in RING-HC domains. In addition, there are seven amino acids between metal ligands 4 and 5 as opposed to two to three in RING-HC and RING-H2 domains. Two of the RING-v domains have a larger number of amino acids between metal ligands 2 and 3 than found previously (Supplemental Fig. 3). Twenty-five predicted RING-v domains were identified in Arabidopsis. The two other modified RING domains previously described in other organisms, RING-G (Dasgupta et al., 2004) and RING-C2 (Albert et al., 2002), have metal ligand substitutions, a glycyl residue for C5 and a cysteinyl residue for H4, respectively (Fig. 1A). Both of these modified RING domain types can be found in the predicted Arabidopsis proteome, with 1 and 10 members, respectively (Table I; Supplemental Table I). The Arabidopsis RING-C2 domains differ from the previous RING-C2 consensus (Albert et al., 2002), with a more variable region between metal ligands 2 and 3, metal ligands 3 and 4, and metal ligands 4 and 5 (Fig. 1A). Two previously unrecognized modified RING domains, RING-D and RING-S/T types, were identified in the Arabidopsis predicted proteome. The RING-D domain present in 10 proteins contains an Asp residue at metal ligand 5, whereas the RING-S/T type in 4 proteins contains either a Ser or Thr at one or both metal ligand positions 2 and 6.
Characteristic of RING domains is the spacing between metal ligand residues, with short distances between all except between metal ligands 2 and 3 and 6 and 7. For all RING types, the most frequent spacing between the amino acids of each zinc-coordinating pair is two between position metal ligands 1 and 2 (100%), one between metal ligands 3 and 4 (96%), two between metal ligands 5 and 6 (99%), and two between metal ligands 7 and 8 (91%; Fig. 1B). The spacing variation observed between the last zinc-coordinating pair, metal ligands 7 and 8, is due to the expanded spacing in the 41 RING-HCb types (with 3–9 amino acids). Only 84% of the RING domains have 2 amino acids between metal ligands 4 and 5 (Fig. 1B). This lowest percentage of conserved number of amino acids is because all 25 RING-v domains have 7 amino acids, the RING-C2 domains differ with 4 or 5 amino acids, and 39 of the RING-HCa domains have 3 amino acids in this region (Fig. 1B; Supplemental Table I).
For each Arabidopsis RING type, the length of the variable loops between metal ligands 2 and 3 and metal ligands 6 and 7 showed some differences compared to the known RING domains (Fig. 1A). For example, the previously published consensus for the RING-H2 domain has 9 to 39 and 4 to 48 amino acids between metal ligands 2 and 3 and metal ligands 6 and 7, respectively (Borden, 2000
Eight proteins were found to contain multiple RING domains (Supplemental Table I). The Arabidopsis ARI proteins, though previously characterized as having two RING domains (Mladek et al., 2003 Several loci with incomplete RING domains in the publicly released annotation were included in this study because analysis of the genomic sequence indicated that the lack of a complete RING domain might be due to an error in the splicing or premature translation stop predictions. No complementary DNA (cDNA) sequences are available for these loci (GenBank, July 2004), and reverse transcription (RT)-PCR experiments failed to amplify products that could be sequenced to determine the endogenous splicing pattern (data not shown). However, gene prediction programs produce a spliced version that is predicted to encode for proteins with complete RING domains with significant similarity to other predicted RING proteins (Supplemental Table III). Conversely, using this same approach, we were unable to detect a complete RING domain at a number of other loci, even though the predicted proteins were related to a particular family of RING proteins (Supplemental Table IV).
BLAST searches of the translated Arabidopsis genome also identified RING domain-encoding sequences in unannotated regions of the genome (Supplemental Table V). RING domains that were previously classified as false positive or as having an incomplete RING were also analyzed (Table I; Supplemental Table I; Kosarev et al., 2002
Except for the RING-D, all other seven types of RING domains identified were also found in the genomes of other organisms. BLAST searches using representative RING-D domains did not retrieve a similar type of domain from any other genome, including other plants such as Oryza sativa. This suggests that this type of modified RING domain is taxonomically more restricted. By contrast, proteins similar to the Arabidopsis RING-S/T-containing proteins (Table II, RING group 15) were found in other species such as yeast, human, and Plasmodium falciparum. However, the RING-S/T domains of many of these proteins were missing metal ligand position 5 or 6 (data not shown). The RING-G domain identified in the yeast San1 (Schnell et al., 1989
Amino acid sequences of representative RING domains from each type are shown in Figure 2. An alignment of all RING domains, except for the RING-v type, can be found in Supplemental Figure 1. The RING-v domains were analyzed separately due to the high sequence similarity within the group and spacing variations (Supplemental Fig. 3). Close examination of the multiple sequence alignment identified a number of amino acids, outside of the metal ligand residues, that are fairly conserved (Figs. 1A and 2). In the majority of RING-H2, RING-HCa/b, RING-v, and RING-D domains, an Ile or Val precedes metal ligand 2. More than 80% of all RING-H2 and RING-D domains have a Phe or Tyr residue in front of metal ligand 5. The Phe is also often present in front of metal ligand 5 of the RING-HCa/b domains. The RING-v domain has an Ala or Val instead of a Phe in front of metal ligand 5. These amino acids are less conserved or rarely found in the RING-C2 domain. In all RING types, except for the RING-HCb and RING-C2, a Trp is usually the fourth amino acid C-terminal to metal ligand 6. Interestingly, the Pro residue present right after metal ligand 7 in all other RING domain types is absent in RING-v proteins. Within the RING-v domain, the amino acid residues observed between metal ligands 7 and 8 are usually Glu followed by Ile or Val. Within the RING-v and RING-D groups, other amino acids also are well conserved (Figs. 1A and 2). Notably, the amino acid after the first metal ligand of the RING-v domain is almost always an Arg. Also, between metal ligand positions 6 and 7, the following amino acids are well conserved: C6-x3-[W]-x3-[KG]-x3-4-C7. A Glu and Ile/Val are observed between metal ligands 7 and 8 in more than 50% and 80% of the RING-v domains, respectively. We also observed that the amino acid immediately following the C8 position of all RING domains is usually a Lys or Arg (data not shown).
To define related groups of RING proteins, we analyzed a phylogenetic tree generated using the various types of RING domains. Domains of similar RING type clustered together, although within each RING type no large clade of related RING domains was observed (Supplemental Fig. 2). The RING-H2, RING-HCa, RING-HCb, RING-C2, and RING-S/T domains were grouped into small clades containing two to eight domains. The RING-D domains form a single distinct clade (Supplemental Fig. 2). The RING domains consistently fell into the same clades after several independent trials of tree generation. The results of the phylogenetic analysis suggest that, though the RING domains share several key features, outside of the conserved metal ligand residues the RING proteins are very distinct. To further define the RING proteins, we examined each protein for the presence of other known domains. Domain predictions resulted in the identification of 30 groups of RING proteins based upon domain presence and organization (Table II; Supplemental Table II). With the exception of a few groups, such as groups 1 (contains no known domain) and 24 (contains predicted transmembrane domain[s]), the number of proteins within each group is small, with some groups containing only one representative. RING groups with larger numbers usually contain proteins encoded by genes that are arranged in tandem repeats. For example, RING group 11.1 contains two sets of tandem repeated genes on chromosomes 2 and 3, containing three and six genes, respectively (Table II; Supplemental Table I). Overall, there are more than 20 groups of tandemly repeated genes, containing two to six loci that encode for RING-containing proteins. Proteins within each distinctive group or subgroup contain the same type of RING domain. For example, all the RING domains of subgroup 27.1 are of the RING-H2 type, and those of subgroup 27.2 are of the RING-HCa type (Table I). The only obvious difference between these two subgroups is the position of the RING domain within the protein (Table II; Supplemental Table II). Even more striking is the fact that the majority of domains of the multiple RING-containing proteins are of the RING-HCa type (Supplemental Table I). For example, PRT1 (At3g24800) contains two RING-HCa domains. The findings from our domain analysis are reflected in the phylogenetic tree, where RING domains of proteins within a particular group tend to clade together (Supplemental Fig. 2). This gives further support to the relationships defined by the RING domain phylogenetic tree. The RING proteins are predicted to contain a surprisingly diverse array of previously described domains. The majority of these are protein-protein interaction domains, which may function as the substrate-binding domain of the E3 ligase (Table II). Predicted protein interaction domains include Ankyrin repeats, BRCT, vWA, coiled-coil domains, and WD40 repeats. Thirteen of the RING groups or subgroups were found only in plant genomes. For example, proteins containing a RING domain in combination with PPR repeats, vWA, or JmjC domains were found only in the Arabidopsis proteome or also in other plant species, such as O. sativa, Medicago truncatula, or Pisum sativum (for domain definitions, see Table II). The most prevalent type of RING proteins are those predicted to contain a transmembrane domain (group 24). Another 29 RING proteins are predicted to contain only a signal peptide (group 25). Three types of zinc fingers were found associated with the RING domain: ZNF_C2H2 (group 29.1), ZNF_C3H1 (group 29.2), and ZNF_ZZ (group 2.2). Both ZNF_C2H2 and ZNF_C3H1 are characterized as nucleic acid-binding zinc fingers. ZNF_ZZ is thought to mediate protein-protein interactions. RING proteins were also found to contain the ubiquitin-binding domains CUE (group 7) or ZNF_UBP (group 29.3). Other domains associated with ubiquitination were also identified. For example, a single DEXDc/RING/HELICc protein (group 8.3) contains an F-box domain, and another was found to contain a PHD domain (group 2.1). The RWD domain, which shows homology to the catalytic domain of UBCs but lacks the conserved Cys required for activity, was also found associated with the RING domain (group 11.2). A significant number of RING proteins did not contain any other detectable, previously described domains (Table II; group 1). Sequence analysis of these RING proteins revealed regions of similarity between a few proteins outside of the RING domain. These putative domains are referred to as DAR1, DAR2, and DAR3 (for domain associated with RING). Representative domains are shown in Figure 3, and a complete list of all putative DAR domains identified is presented in Supplemental Figure 4. DAR1 and DAR3 are approximately 40 amino acids in length, and DAR2 consists of more than 120 amino acids. BLAST searches with the putative DAR domains retrieved predicted proteins from other species with similar regions (Fig. 3; Supplemental Fig. 4). DAR1 and DAR3 were found only in O. sativa and/or Lotus japonicus predicted proteins, whereas DAR2 was found in predicted proteins from a number of genomes, including human, Caenorhabditis elegans, D. melanogaster, and O. sativa. A number of the DAR3-containing Arabidopsis proteins show a low level of similarity to CIP8 (data not shown). In almost all proteins, the putative DAR domains were found associated with a RING domain. In these cases, the DAR domain was found N-terminal to the RING domain.
Arabidopsis RING Domains Possess E3 Ubiquitin Ligase Activity
RING proteins are involved in numerous cellular processes including transcription, signal transduction, and recombination. Functions attributed to the RING domain itself include protein-protein interaction and more recently protein ubiquitination (Lorick et al., 1999
We tested the biochemical activity of representative full-length proteins from each RING and modified RING type. GST-tagged RING-H2, RING-HCa, RING-HCb, RING-v, RING-C2, and RING-D domain-containing proteins were all found to possess E2-dependent E3 ligase activity in vitro, whereas we were unable to detect ligase activity for the RING-S/T domain-containing proteins (Fig. 4; Table I). Twenty-two of the 29 GST-RING-H2 proteins analyzed were active in the in vitro assay. Of the 19 RING-HCa types tested, polyubiquitination was not observed for 8 (Fig. 4; Table I). RING-HCb protein At1g65430/ARI8 was active in the in vitro assay. However, no polyubiquitination was observed for the other RING-HCb protein, At3g45555, tested. Two of the four RING-S/T proteins identified were tested, and no E3 ubiquitin ligase activity was observed for either GST-RING-S/T protein (Table I). Of the six GST-RING-v proteins tested, all were capable of promoting polyubiquitination (Fig. 4; Table I). Four RING-D proteins were chosen for biochemical analysis. Ubiquitin ligase activity was observed for three of the four GST-RING-D proteins (Fig. 4; Table I). Both GST-RING-C2 proteins analyzed were active in the in vitro ubiquitination assay (Fig. 4). The RING-G-containing protein was not analyzed. However, the RING-G-containing San1 protein from yeast has been shown to mediate protein ubiquitination in vitro (Dasgupta et al., 2004
To test the requirement of the RING and modified RING domains for ubiquitin ligase activity, well-conserved amino acids in the RING domains of selected proteins were mutated and the mutated proteins assayed for ubiquitination activity. Mutation of one or more of the metal ligands has been shown previously to disrupt the ability of the RING domain to promote protein ubiquitination (Lorick et al., 1999
Previous analysis of the RING-containing protein family reported that the Arabidopsis genome encodes for 387 RING and modified RING domains (Kosarev et al., 2002 ). Our analysis identified 90 additional RING domains. Therefore, the Arabidopsis genome contains at least 469 genes encoding for RING domain-containing proteins. The RING genes account for approximately 2% of the predicted protein-encoding genes of Arabidopsis. Unlike the F-box family, which contains twice as many proteins, the size of the Arabidopsis RING family is comparable to the numbers found in other eukaryotes such as human and Mus musculus, which contain 385 and 305 RING domains, respectively (Semple, 2003). The D. melanogaster and C. elegans genomes are predicted to encode for 121 and 170 RING proteins, respectively, and the yeast genome encodes for 14. Although many RING proteins are unique, the number of RING domains encoded by tandemly repeated genes suggests that the size of the Arabidopsis RING family may be due in part to gene duplication. Therefore, the RING family contains both unique and tandemly duplicated members. However, functional studies could uncover unique roles for closely linked genes.
A variety of protein-protein interaction domains, ubiquitin and nucleic acid-binding domains, and a diverse array of domains with unknown function were found associated with the RING domain. A number of these domains are also found in functional RING E3 ligases from other eukaryotes. For example, the domain organization of the Arabidopsis CUE-containing RING proteins is similar to the mammalian transmembrane receptor gp78, an endoplasmic reticulum E3 ligase (Fang et al., 2001
The domain organization of a number of RING-containing proteins is plant-specific. The Znf_ZZ-containing multiple RING domain-containing E3 ligase protein PRT1, which is involved in the N-end rule ubiquitination pathway, is only detected in the Arabidopsis genome (Potuschak et al., 1998
Almost 500 proteins representing 8 types of RING domains were identified. Five of the eight types identified are modified RING domains that display variation in spacing between, or have amino acids substitutions at, conserved zinc-coordinating residues. Biochemical analysis of representative RING domain-containing proteins from each RING type demonstrates that certain modifications do not affect E3 ligase activity in vitro, whereas others seem to abrogate E3 activity. As shown here and in previous studies, RING domains utilizing a Cys at all metal ligand positions (RING-C2) and domains with spacing variations (RING-v) are functional E3 ligase domains (Albert et al., 2002 The RING-S/T-containing proteins were not functional in our in vitro assays, indicating that Ser or Thr substitutions at metal ligand position 2 or 6 are not acceptable, at least in the context of this protein in combination with AtUBC8. We also failed to detect activity for the RING-S/T proteins in in vitro assays with other AtUBCs, such as AtUBC10 and AtUBC11, which are similar to AtUBC8, and AtUBC35 and AtUBC36 (S. Stone, E. Kraft, and J. Callis, unpublished data). The RING-S/T domains of the LisH and CTLH protein family may not represent a bona fide RING domain, or the RING may have lost the ability to function as an ubiquitin ligase domain. However, not all UBCs have been tested, and these RING domains could function with one specific E2. Alternatively, RING-S/T domains could require an additional protein/cofactor/modification that is not present in our in vitro assays. Similarly, a number of RING-H2- and RING-HCa/b-containing proteins analyzed also showed no activity in the biochemical assay. To function as E3 ligases in vitro, these proteins may also require cofactors or a specific type of E2 to facilitate protein ubiquitination. Therefore, we cannot dismiss these RING or modified RING proteins as nonfunctional or as not containing a bona fide RING domain.
In addition to the eight zinc-coordinating amino acids, other residues are important for the RING domain to function as an E3 ligase. For many RING domains, the amino acid preceding metal ligand position 2 is usually a hydrophobic amino acid, such as Leu, Ile, or Val. This hydrophobic amino acid at this position, located within the binding site of the first zinc ion, is utilized by E3 ligases CNOT4 (RING-C2), c-CBL (RING-HCa), and BRCA1 (RING-HCa) during E2 binding and is required for E3 activity, as mutation of this amino acid abrogates activity (Zheng et al., 2000
The information provided in this study demonstrates that the Arabidopsis RING-type family of E3 ligases is quite diverse, and, in addition to the previously defined types of RING domain, Arabidopsis utilizes a number of variations on the canonical RING domain. Therefore, searches for RING domain-containing proteins should not be limited to the known types. However, more characterization of the Arabidopsis RING types is needed so as to further define the requirements for a functional RING domain. The presence of the various types of RING domains found may represent another level of specificity within the ubiquitination pathway, in which each type of RING domain may function in vivo with a particular subset of the more than 37 Arabidopsis E2 enzymes (Bachmair et al., 2001
Identification of Arabidopsis RING Domain-Containing Proteins InterPro (Integrated Documentation Resource of Protein Families, release v7.2, March 29, 2004; http://www.ebi.ac.uk/interpro/) database was used to retrieve Arabidopsis (Arabidopsis thaliana) proteins containing a RING domain [InterPro domain IPR001841 (Znf_RING); Prosite domain PS00518 (ZF_RING_1); Pfam domain PF00097 (zf-C3HC4); SMART domain SM00184 (RING)]. RING domains of known Arabidopsis and mammalian RING proteins were used in BLAST searches against the complete nonredundant Arabidopsis genome (TAIR, April 16, 2003; http://www.arabidopsis.org). Retrieved sequences were analyzed by the SMART database (version 4.0, May 28, 2004; http://smart.embl-heidelberg.de/), followed by manual inspection. Each potential RING domain was analyzed manually for the presence of, and distance between, each of the eight zinc-coordinating Cys and/or His residues.
Database search for RING domains also retrieved proteins with related Cys-rich domains, such as the PHD (C4HC3) and LIM (C2HC4C/H; Capili et al., 2001
The ClustalX program using the PAM350 protein matrix, with gap opening and gap extension penalty parameters of 35.0 and 0.75, respectively, in pairwise alignment and 15.0 and 0.3, respectively, in the multiple alignments was used to generate the initial alignment of RING domains. Se-Al sequence editor (Evolutionary Biology Group, University of Oxford, UK) was used to manually edit the alignment. Unrooted phylogenetic trees were created by PAUP* (Phylogenetic Analysis Using Parsimony) version 4.0 using the neighbor-joining method with 1,000 bootstrap replicates. Gene annotation database GENSCAN (http://genes.mit.edu/GENSCAN.html) and/or manual editing were used to analyzed genomic sequences to predict alternate open reading frames (ORFs) for potentially misannotated genes. For domain identification, SMART and Pfam (Protein families database of alignments and HMMs, version 14.0, June 2004; http://www.sanger.ac.uk/Software/Pfam/) databases were used to analyze each protein sequence. To identify potential novel domains, sequence alignments and BLAST searches were performed using the entire amino acid sequence of each RING protein. Phylogenetic analysis was used to confirm the amino acid sequence similarity observed between proteins.
Seeds from Arabidopsis ecotype Col-0 were either sown on soil and grown under photoperiodic cycles of 16 h light and 8 h dark at 16°C with 50% relative humidity, or seeds surface sterilized with 30% (v/v) bleach and 0.1% (v/v) Triton X-100 were grown on 1% (w/v) agar with 1x Murashige and Skoog and 1% (w/v) Suc under continuous light.
Total RNA isolated from Arabidopsis ecotype Col-0, 10-d-old seedlings, leaves (2- to 4-week-old plants), or a mixture of RNA from leaves, seedlings, and flowers (6- to 7-week-old plants) was used in reverse-transcription reactions followed by PCRs to amplify the complete predicted ORF for each RING gene with one or more introns. The Qiagen RNeasy plant RNA extraction kit (Qiagen, Valencia, CA) was used to isolate total RNA as per the manufacturer's instructions. For intronless genes, the complete ORF was isolated from leaf (2- to 4-week-old plants) DNA. Otherwise, the ORF was isolated from expressed sequence tag (EST) clones obtained from the Arabidopsis Biological Resource Center (ABRC). The source of RNA/DNA used to isolate the ORF for each particular gene is outlined in Table I. The Gateway cloning system (Invitrogen, Carlsbad, CA) was used to clone each RING gene. Full-length RING cDNAs were first introduced into the Gateway entry vector pDONR and the DNA sequence determined. Sequences of each RING cDNA were compared with the predicted ORF available on the TAIR (http://www.arabidopsis.org) and The Institute for Genomic Research (http://www.tigr.org) Arabidopsis genome annotation databases. cDNAs matching the predicted ORFs were introduced into the pDEST15 (Invitrogen) protein expression vector to produce in-frame fusions with the GST tag. AtUBC8 was cloned in a similar manner and introduced into the pDEST17 (Invitrogen) vector to produce an in-frame fusion with the 6xHis tag. For RING mutational analysis, site-directed mutagenesis (Stratagene, La Jolla, CA) was used to make a series of point mutations within the region of the gene predicted to encode for the RING domain. pDONR vector containing the RING cDNA of interest was used as a PCR template. Sequence analysis was used to confirm nucleotide changes. The mutated RING cDNA was introduced, via the Gateway system, into the pDEST15 vector to produce an in-frame fusion with the GST tag.
GST-RING or mutated RING fusions were expressed in Escherichia coli strain BL21 (DE3) pLysS in 50-mL cultures. Transformed cells were grown at 37°C for 2 to 3 h or to an OD600 of 0.4 to 0.6 before induction with 0.5 mM isopropylthio- SDS-PAGE electrophoresis followed by Coomassie blue staining and Bradford assays (Bio-Rad, Hercules, CA) were used to quantify purified proteins. Western-blot analysis using GST antibodies or His antibodies (Amersham, Buckinghamshire, UK) was also used to confirm the presence and integrity of each fusion protein.
Ubiquitination assays were carried out as described previously (Hardtke et al., 2002 Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor.
We thank M. Wogulis (University of California, Davis) for the gift of E1 protein and helpful discussions, and members of the Callis lab for helpful discussions and editing. We thank Dr. Richard Gardner (Fred Hutchinson Cancer Research Center, Seattle) for helpful information regarding the Arabidopsis RING-G protein. We would also like to thank Charles Forsyth and Colin Wu (University of California, Davis) for assistance in the assembly of the RING database. Received August 25, 2004; returned for revision November 2, 2004; accepted November 10, 2004.
1 This work was supported by the National Science Foundation (2010 grant no. MCB–00115870). E.K. also was partially supported by the National Institute of Health Training (grant no. GM0007377–27), and S.L.S. was supported by the Natural Sciences and Engineering Research Council of Canada and the International Human Frontier Science Program fellowships. 
2 Present address: Molecular Biology Laboratory, Institute for Experimental Pathology, University of Iceland, Keldur, v/Vesturlandsveg, IS-112 Reykjavik, Iceland.
3 Present address: Laboratory for Molecular and Computational Genomics, UW Biotechnology Center, Madison, WI 53706.
[w] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.104.052423. * Corresponding author; e-mail jcallis{at}ucdavis.edu; fax 530–752–3085.
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ABSTRACT
RESULTS
80% of domains, and blue and green in 
50% of domains, respectively. X(n) indicates number of spacing amino acids between conserved amino acids. B, Spacing variation observed between zinc-coordinating metal ligand (ml) pairs in Arabidopsis RING domains. Number of amino acids between each metal ligand and the number of domains in which the spacing variation is found are shown. *, Spacing unique to RING-HCa protein At2g42160, metal ligand position 5 assignment uncertain. C, Comparison of the number of amino acids in the loops between metal ligands 2 and 3 and metal ligands 6 and 7 of RING-H2, RING-HCa, and RING-HCb domains.
-galactoside for 3 to 4 h at 37°C. Cells were harvested by centrifugation and lysed in 2 mL of lysis buffer containing 25 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.01% Triton X-100. For purification, 100 µL of glutathione agarose beads (Sigma, St. Louis) was added to cleared lysates and incubated for 2 h at 4°C. Beads were then washed four times with 1 mL of wash buffer containing 25 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 0.01% (v/v) Triton X-100. GST fusion proteins were eluted with 100 µL of elution buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.01% (v/v) Triton X-100 supplemented with 15 mM reduced glutathione, or the GST fusion proteins were left bound to the beads. Forty microliters of glycerol was added to the eluted protein. Bead-bound GST proteins were stored in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% (v/v) Triton X-100, and 40% (v/v) glycerol. Proteins were stored at –80°C until needed. The 6xHis-tagged AtUBC8 was expressed and purified in a similar manner. The 6xHis fusion was expressed in E. coli strain BL21 AI (Invitrogen) and induced with 0.2% (w/v) Ara. Lysis and wash buffer were supplemented with 5 mM imidazole and elution buffer supplemented with 300 mM imidazole.