Sophia L. Stone

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.

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.

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.

ARC1 Is an E3 Ubiquitin Ligase and Promotes the Ubiquitination of Proteins during the Rejection of Self-Incompatible Brassica Pollen

ARC1 is a novel U-box protein required in the Brassica pistil for the rejection of self-incompatible pollen; it functions downstream of the S receptor kinase (SRK). Here, we show that ARC1 has E3 ubiquitin ligase activity and contains several motifs that influence its subcellular localization. ARC1 can shuttle between the nucleus, cytosol, and proteasome/COP9 signalosome (CSN) when expressed in tobacco BY-2 suspension-cultured cells. However, ARC1 localization to the proteasome/CSN occurs only in the presence of an active SRK. In the pistil, ubiquitinated protein levels increase specifically with incompatible pollinations, but they do not change in ARC1 antisense-suppressed pistils. In addition, inhibition of the proteasomal proteolytic activity disrupts the self-incompatibility response. We propose that ARC1 promotes the ubiquitination and proteasomal degradation of compatibility factors in the pistil, which in turn leads to pollen rejection.

Abiotic stress tolerance mediated by protein ubiquitination

Plant growth and development is largely influenced by ubiquitin-mediated regulation of protein stability. Specificity of the ubiquitination pathway is controlled mainly by the substrate-recruiting E3 ubiquitin ligases, and consequently, E3 ligases control numerous cellular processes. Recent evidence that ubiquitination plays a critical role in regulating plant responses to abiotic stresses has launched intensive efforts to identify E3 ligases that mediate plant tolerance of adverse environmental conditions. Most stress-related E3 ligases identified to date facilitate responses to environmental stimuli by modulating the abundance of key downstream stress-responsive transcription factors. In this review, the regulatory roles of ubiquitin during the plants response to abiotic stress are summarized and highlighted.

Cellular Pathways Regulating Responses to Compatible and Self-Incompatible Pollen in Brassica and Arabidopsis Stigmas Intersect at Exo70A1, a Putative Component of the Exocyst Complex

In the Brassicaceae, compatible pollen–pistil interactions result in pollen adhesion to the stigma, while pollen grains from unrelated plant species are largely ignored. There can also be an additional layer of recognition to prevent self-fertilization, the self-incompatibility response, whereby self pollen grains are distinguished from nonself pollen grains and rejected. This pathway is activated in the stigma and involves the ARM repeat–containing 1 (ARC1) protein, an E3 ubiquitin ligase. In a screen for ARC1-interacting proteins, we have identified Brassica napus Exo70A1, a putative component of the exocyst complex that is known to regulate polarized secretion. We show through transgenic studies that loss of Exo70A1 in Brassica and Arabidopsis thaliana stigmas leads to the rejection of compatible pollen at the same stage as the self-incompatibility response. A red fluorescent protein:Exo70A1 fusion rescues this stigmatic defect in Arabidopsis and is found to be mobilized to the plasma membrane concomitant with flowers opening. By contrast, increased expression of Exo70A1 in self-incompatible Brassica partially overcomes the self pollen rejection response. Thus, our data show that the Exo70A1 protein functions at the intersection of two cellular pathways, where it is required in the stigma for the acceptance of compatible pollen in both Brassica and Arabidopsis and is negatively regulated by Brassica self-incompatibility.

Abscisic Acid Increases Arabidopsis ABI5 Transcription Factor Levels by Promoting KEG E3 Ligase Self-Ubiquitination and Proteasomal Degradation

This work shows that KEG E3 ligase activity is required for regulation of the abundance of the ABA responsive transcription factor ABI5. It shows that KEG undergoes autoubiquitination in response to ABA and is degraded by the 26S proteasome, allowing ABI5 levels to increase and mediate cellular ABA responses. The Arabidopsis thaliana RING-type E3 ligase KEEP ON GOING (KEG) is a negative regulator of abscisic acid (ABA) signaling. Seedlings homozygous for T-DNA insertions in KEG accumulate high levels of the ABA-responsive transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5). Here, we demonstrate that KEG E3 ligase activity is required for the regulation of ABI5 abundance. KEG ubiquitinates ABI5 in vitro, and a functional KEG RING domain is required to restore the levels of ABI5 in keg-1 to that of the wild type. Overexpression of KEG leads to ABA insensitivity, which correlates with KEG protein levels. In the presence of ABA, ABI5 levels increase drastically via a decrease in ubiquitin-meditated proteasomal degradation. Our results indicate that ABA promotes ABI5 accumulation by inducing the ubiquitination and proteasomal degradation of KEG. A functional RING domain is required for the ABA-induced degradation of KEG, suggesting that the loss is due to self-ubiquitination. Mutations within KEGs kinase domain or treatments with kinase inhibitors prohibit the ABA-induced ubiquitination and degradation of KEG, indicating that phosphorylation, possibly self-phosphorylation, is involved in the ABA regulation of KEG protein levels. We discuss a model for how ABA may negatively regulate KEG protein abundance, leading to accumulation of ABI5 and ABA-dependent cellular responses.

A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.

The role of ubiquitin and the 26S proteasome in plant abiotic stress signaling

Ubiquitin is a small, highly conserved, ubiquitously expressed eukaryotic protein with immensely important and diverse regulatory functions. A well-studied function of ubiquitin is its role in selective proteolysis by the ubiquitin-proteasome system (UPS). The UPS has emerged as an integral player in plant response and adaptation to environmental stresses such as drought, salinity, cold and nutrient deprivation. The UPS has also been shown to influence the production and signal transduction of stress-related hormones such as abscisic acid. Understanding UPS function has centered mainly on defining the role of E3 ubiquitin ligases, which are the substrate-recruiting component of the ubiquitination pathway. The recent identification of stress signaling/regulatory proteins that are the subject of ubiquitin-dependent degradation has increased our knowledge of how the UPS facilitates responses to adverse environmental conditions. A brief overview is provided on role of the UPS in modulating protein stability during abiotic stress signaling. E3 ubiquitin ligases for which stress-related substrate proteins have been identified are discussed.

Arabidopsis CIPK26 interacts with KEG, components of the ABA signalling network and is degraded by the ubiquitin-proteasome system.

The RING-type E3 ligase, Keep on Going (KEG), is required for early seedling establishment in Arabidopsis thaliana. Post-germination, KEG negatively regulates abscisic acid (ABA) signalling by targeting Abscisic Acid Insensitive 5 (ABI5) for ubiquitination and subsequent degradation. Previous reports suggest that the role of KEG during early seedling development is not limited to regulation of ABI5 abundance. Using a yeast two-hybrid screen, this study identified Calcineurin B-like Interacting Protein Kinase (CIPK) 26 as a KEG-interacting protein. In vitro pull-down and in planta bimolecular fluorescence complementation assays confirmed the interactions between CIPK26 and KEG. In planta experiments demonstrated that CIPK26 was ubiquitinated and degraded via the 26S proteasome. It was also found that turnover of CIPK26 was increased when KEG protein levels were elevated, suggesting that the RING-type E3 ligase is involved in targeting CIPK26 for degradation. CIPK26 was found to interact with the ABA signalling components ABI1, ABI2, and ABI5. In addition, CIPK26 was capable of phosphorylating ABI5 in vitro. Consistent with a role in ABA signalling, overexpression of CIPK26 increased the sensitivity of germinating seeds to the inhibitory effects of ABA. The data presented in this report suggest that KEG mediates the proteasomal degradation of CIPK26 and that CIPK26 is part of the ABA signalling network.