Judy Callis

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

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 SRA Methyl-Cytosine-Binding Domain Links DNA and Histone Methylation

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.

KEEP ON GOING, a RING E3 Ligase Essential for Arabidopsis Growth and Development, Is Involved in Abscisic Acid Signaling

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.

Regulation of Protein Degradation.

of understanding of how to preserve in vivo proteolytic activities in vitro also has interfered with the identification, purification, and analyses of proteinases. Although studies using heterologous proteins or synthetic peptide substrates will continue to provide valuable information, such studies must at some point utilize in vivo substrates if the physiological roles of proteinases are to be defined. Finally, no particular global feature of proteins can be correlated with in vivo stability, again indicating the diversity of mechanisms present. Proteins may be subjected to a limited number of proteolytic cleavages either in the process of intracellular targeting to their final destination or in the maturation of the initial translation product to produce a biologically active mature molecule. This review does not discuss these events but rather focuses on what is known about the events that regulate, orare responsible for, the initial cleavages of a protein ultimately destined for hydrolysis to free amino acids. Our current understanding of the protein degradation process is that it occurs in stages, with the initial endoproteolytic cleavage(s) being more species specific and rate limiting than subsequent hydrolysis to free amino acids by less specific endo- and exopeptidases. For this reason, this review limits discussion to endoproteinases, which are referred to here simply as proteinases. Recent advances in our understanding of the regulation of protein degradation have been made on two fronts: first, in the identification of proteinases, changes in whose activity and/or abundance could or do result in the degradation of previously stable protein(s); and second, in the identification of structural changes in proteins that increase their susceptibility to proteolysis. Advances in these two areas made after a previous review on plant proteolysis (Vierstra, 1993) are elaborated in the following discussions. Recently, multiple cases have been documented in which increases in the levels of mRNAs encoding proteinases occur in response to developmental or environmental changes. The physiological significance of such increases remains unknown; however, they indicate physiological responses that may be mediated by proteolysis or that result in accelerated proteolysis and are therefore mentioned here. Cell cycle progression in eukaryotes represents an exquisite example of the importance of regulated protein degradation, and recent data regarding proteolysis and cell cycle transitions are discussed. A brief description of the diversity of proteinases present in plants is presented, focusing on recent work on

Genome Analysis and Functional Characterization of the E2 and RING-Type E3 Ligase Ubiquitination Enzymes of Arabidopsis

Attachment of ubiquitin to substrate proteins is catalyzed by the three enzymes E1, E2 (ubiquitin conjugating [UBC]), and E3 (ubiquitin ligase). Forty-one functional proteins with a UBC domain and active-site cysteine are predicted in the Arabidopsis (Arabidopsis thaliana) genome, which includes four that are predicted or shown to function with ubiquitin-like proteins. Only nine were previously characterized biochemically as ubiquitin E2s. We obtained soluble protein for 22 of the 28 uncharacterized UBCs after expression in Escherichia coli and demonstrated that 16 function as ubiquitin E2s. Twelve, plus three previously characterized ubiquitin E2s, were also tested for the ability to catalyze ubiquitination in vitro in the presence of one of 65 really interesting new gene (RING) E3 ligases. UBC22, UBC19-20, and UBC1-6 had variable levels of E3-independent activity. Six UBCs were inactive with all RINGs tested. Closely related UBC8, 10, 11, and 28 were active with the largest number of RING E3s and with all RING types. Expression analysis was performed to determine whether E2s or E3s were expressed in specific organs or under specific environmental conditions. Closely related E2s show unique patterns of expression and most express ubiquitously. Some RING E3s are also ubiquitously expressed; however, others show organ-specific expression. Of all the organs tested, RING mRNAs are most abundant in floral organs. This study demonstrates that E2 diversity includes examples with broad and narrow specificity toward RINGs, and that most ubiquitin E2s are broadly expressed with each having a unique spatial and developmental pattern of expression.

The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression.

We have isolated and determined DNA sequence for the 5′-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5′-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5′-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5′-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli β-glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5′ intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.

The Arabidopsis Aux/IAA Protein Family Has Diversified in Degradation and Auxin Responsiveness

Rapid, auxin-responsive degradation of multiple auxin/indole-3-acetic acid (Aux/IAA) proteins is essential for plant growth and development. Domain II residues were previously shown to be required for the degradation of several Arabidopsis thaliana Aux/IAA proteins. We examined the degradation of additional full-length family members and the proteolytic importance of N-terminal residues outside domain II using luciferase (LUC) fusions. Elimination of domain I did not affect degradation. However, substituting an Arg for a conserved Lys between domains I and II specifically impaired basal degradation without compromising the auxin-mediated acceleration of degradation. IAA8, IAA9, and IAA28 contain domain II and a conserved Lys, but they were degraded more slowly than previously characterized family members when expressed as LUC fusions, suggesting that sequences outside domain II influence proteolysis. We analyzed the degradation of IAA31, with a region somewhat similar to domain II but without the conserved Lys, and of IAA20, which lacks domain II and the conserved Lys. Both IAA20:LUC and epitope-tagged IAA20 were long-lived, and their longevity was not influenced by auxin. Epitope-tagged IAA31 was long-lived, like IAA20, but by contrast, it showed accelerated degradation in response to auxin. The existence of long-lived and auxin-insensitive Aux/IAA proteins suggeststhat they may play a novel role in auxin signaling.

Auxin modulates the degradation rate of Aux/IAA proteins

Aux/IAA gene family members were first identified by their rapid transcriptional increase in response to auxin. Auxin/indole-3-acetic acid protein (Aux/IAA) luciferase (LUC) fusions expressed in Arabidopsis under control of a non-auxin-responsive promoter were used to monitor the effect of auxin on protein abundance independent of transcriptional regulation by auxin. After 2 hr in the presence of 1 μM exogenous dichlorophenoxyacetic acid (2,4D), a synthetic auxin, the levels of pea IAA6 (PSIAA6) and Arabidopsis IAA1 LUC activity were 35% and 67%, respectively, of mock-treated genetically identical seedlings, whereas the activity of LUC alone from equivalently treated seedlings remained unaltered. The steady-state level of an Aux/IAA fusion protein lacking domain II, one of the conserved domains found in all Aux/IAA proteins, was not reduced in the presence of auxin. Higher levels of exogenous auxin were required to affect the steady-state level of the PSIAA6∷LUC fusion with a point mutation in domain II. A 13-aa consensus sequence from domain II fused to LUC created an auxin-responsive fusion protein. The change in steady-state levels in response to auxin is extremely rapid, with a decrease in LUC activity detectable by 2 min after auxin application. Direct half-life measurements show that the decrease caused by exogenous auxin is due to the decrease in fusion protein half-life. These results suggest that auxin rapidly modulates the degradation rate of Aux/IAA proteins, with higher levels of auxin increasing the proteolytic rate of Aux/IAA family members.

Protein degradation in signaling

Recent studies have linked proteolysis by the ubiquitin/proteasome pathway to a variety of signaling pathways in higher plants. These links were uncovered by characterization of mutants altered in signaling or by targeted disruption of components of the proteolytic pathway. Significant advances have recently revealed connections between proteolysis and hormone responses, light perception, environmental adaptation, and floral development.

Regulation of Cullin RING Ligases

The ubiquitin/26S proteasome pathway largely mediates selective proteolysis in the nucleus and cytosol. This pathway catalyzes covalent attachment of ubiquitin (UBQ) to substrate proteins in an E1-E2-E3 cascade. Ubiquitin E3 ligases interact with substrates to catalyze UBQ transfer from E2 to substrate. Within the E3 ligase superfamily, cullin RING ligases (CRLs) are significant in plants because they are linked to hormonal signaling, developmental programs, and environmental responses. Thus, knowledge of CRL regulation is required for a complete understanding of these processes. A major mechanism modulating CRL activity is modification of the cullin subunit by RUB (RELATED TO UBIQUITIN), a ubiquitin-like protein, and demodification by the COP9 signalosome (CSN). CULLIN-ASSOCIATED NEDD8-DISSOCIATED 1 (CAND1) interacts with CRLs, affecting both rubylation and derubylation. Described here are the pathways, regulation, and biological function of rubylation and derubylation, as well as future directions and outstanding questions.