AXL and AXR1 have redundant functions in RUB conjugation and growth and development in Arabidopsis

Cullin-RING ubiquitin-protein ligases such as the Skp1, cullin, F-box protein (SCF) have been implicated in many growth and developmental processes in plants. Normal SCF function requires that the CUL1 subunit be post-translationally modified by related to ubiquitin (RUB), a protein related to ubiquitin. This process is mediated by two enzymes: the RUB-activating and RUB-conjugating enzymes. In Arabidopsis, the RUB-activating enzyme is a heterodimer consisting of AXR1 and ECR1. Mutations in the AXR1 gene result in a pleiotropic phenotype that includes resistance to the plant hormone auxin. Here we report that the AXL (AXR1-like) gene also functions in the RUB conjugation pathway. Overexpression of AXL in the axr1-3 background complements the axr1-3 phenotype. Biochemical analysis indicates that AXL overexpression restores CUL1 modification to the wild-type level, indicating that AXR1 and AXL have the same biochemical activity. Although the axl mutant resembles wild-type plants, the majority of axr1 axl-1 double mutants are embryo or seedling lethal. Furthermore, the axl-1 mutation reveals novel RUB-dependent processes in embryo development. We conclude that AXR1 and AXL function redundantly in the RUB conjugating pathway.     

Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function

The ubiquitin-related protein RUB/Nedd8 is conjugated to members of the cullin family of proteins in plants, animals, and fungi. In Arabidopsis, the RUB conjugation pathway consists of a heterodimeric E1 (AXR1-ECR1) and a RUB-E2 called RCE1. The cullin CUL1 is a subunit in SCF-type ubiquitin protein ligases (E3s), including the SCF(TIR1) complex, which is required for response to the plant hormone auxin. Our previous studies showed that conjugation of RUB to CUL1 is required for normal SCF(TIR1) function. The RING-H2 finger protein RBX1 is a subunit of SCF complexes in fungi and animals. The function of RBX1 is to bind the ubiquitin-conjugating enzyme E2 and bring it into close proximity with the E3 substrate. We have identified two Arabidopsis genes encoding RING-H2 proteins related to human RBX1. Studies of one of these proteins indicate that, as in animals and fungi, Arabidopsis RBX1 is an SCF subunit. Reduced RBX1 levels result in severe defects in growth and development. Overexpression of RBX1 increases RUB modification of CUL1. This effect is associated with reduced auxin response and severe growth defects similar to those observed in axr1 mutants. As in the axr1 mutants, RBX1 overexpression stabilizes the SCF(TIR1) substrate AXR2/IAA7. The RBX1 protein is a component of SCF complexes in Arabidopsis. In addition to its direct role in SCF E3 ligase activity, RBX1 promotes the RUB modification of CUL1 and probably functions as an E3 ligase in the RUB pathway. Hypermodification of CUL1 disrupts SCF(TIR1) function, suggesting that cycles of RUB conjugation and removal are important for SCF activity.     

AXR1-ECR1-dependent conjugation of RUB1 to the Arabidopsis Cullin AtCUL1 is required for auxin response

Mutations in the AXR1 gene result in a reduction in auxin response and diverse defects in auxin-regulated growth and development. In a previous study, we showed that AXR1 forms a heterodimer with the ECR1 protein. This enzyme activates the ubiquitin-related protein RUB1 in vitro. Furthermore, we showed that the Skp1-Cul1/Cdc53-F-box (SCF) subunit AtCUL1 is modified by RUB1 in vivo. In this report, we demonstrate that the formation of RUB-AtCUL1 is dependent on AXR1 and ECR1 in vivo. The expression of AXR1 and ECR1 is restricted to zones of active cell division and cell elongation, consistent with their role in growth regulation. These results provide strong support for a model in which RUB conjugation of AtCUL1 affects the function of SCF E3s that are required for auxin response.     

AXR1 promotes the Arabidopsis cytokinin response by facilitating ARR5 proteolysis

The plant hormone cytokinin plays essential roles in many aspects of growth and development. The cytokinin signal is transmitted by a multi‐step phosphorelay to the members of two functionally antagonistic classes of Arabidopsis response regulators (ARRs): type B ARRs (response activators) and type A ARRs (negative‐feedback regulators). Previous studies have shown that mutations in AXR1, encoding a subunit of the E1 enzyme in the RUB (related to ubiquitin) modification pathway, lead to decreased cytokinin sensitivity. Here we show that the cytokinin resistance of axr1 seedlings is suppressed by loss of function of the type A ARR family member ARR5. Based on the established role of the RUB pathway in ubiquitin‐dependent proteolysis, these data suggest that AXR1 promotes the cytokinin response by facilitating type A ARR degradation. Indeed, both genetic (axr1 mutants) and chemical (MLN4924) suppression of RUB E1 increased ARR5 stability, suggesting that the ubiquitin ligase that promotes ARR5 proteolysis requires RUB modification for optimal activity.     

The RUB/Nedd8 conjugation pathway is required for early development in Arabidopsis

The related-to-ubiquitin (RUB) protein is post-translationally conjugated to the cullin subunit of the SCF (SKP1, Cullin, F-box) class of ubiquitin protein ligases. Although the precise biochemical function of RUB modification is unclear, studies indicate that the modification is important for SCF function. In Arabidopsis, RUB modification of CUL1 is required for normal function of SCF(TIR1), an E3 required for response to the plant hormone auxin. In this report we show that an Arabidopsis protein called RCE1 functions as a RUB-conjugating enzyme in vivo. A mutation in the RCE1 gene results in a phenotype like that of the axr1 mutant. Most strikingly, plants deficient in both RCE1 and AXR1 have an embryonic phenotype similar to mp and bdl mutants, previously shown to be deficient in auxin signaling. Based on these results, we suggest that the RUB-conjugation pathway is required for auxin-dependent pattern formation in the developing embryo. In addition, we show that RCE1 interacts directly with the RING protein RBX1 and is present in a stable complex with SCF. We propose that RBX1 functions as an E3 for RUB modification of CUL1.     

Function of the ubiquitin–proteosome pathway in auxin response

Proteolysis of important regulatory proteins by the ubiquitin–proteosome pathway is a key aspect of cellular regulation in eukaryotes. Genetic studies in Arabidopsis indicate that response to auxin depends on the function of proteins in this pathway. The auxin transport inhibitor resistant 1 (TIR1) protein is part of a ubiquitin–protein–ligase complex (E3), known as SKP1 CDC53 F-box^TIR1 (SCF^TIR1), that possibly directs ubiquitin-modification of protein regulators of the auxin response. In yeast, a similar E3 complex, SCF^CDC4, is regulated by conjugation of the ubiquitin-related protein Rub1 to the Cdc53 protein. In Arabidopsis, the auxin-resistant1 (AXR1) gene encodes a subunit of the RUB1-activating enzyme, the first enzyme in the RUB-conjugation pathway. Loss of AXR1 results in loss of auxin response. These results suggest a model in which RUB1 modification regulates the activity of SCF^TIR1, thereby directing the degradation of the repressors of the auxin response.     

Mutation of E1-CONJUGATING ENZYME-RELATED1 decreases RELATED TO UBIQUITIN conjugation and alters auxin response and development

The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.     

Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/NEDD8-activating enzyme in Arabidopsis seedlings

SMALL ACIDIC PROTEIN 1 (SMAP1) functions upstream of the degradation of AUX/IAA-proteins in the response to 2,4-dichlorophenoxyacetic acid and physically interacts with the COP9 SIGNALOSOME (CSN). Also, its function is linked to RELATED TO UBIQUITIN (RUB) modification. To further investigate the relationship between SMAP1 and the RUB modification system, we examined the effect of MLN4924, an inhibitor of RUB/NEDD8-activating E1 enzyme, on the growth of Arabidopsis thaliana. We found that the anti-auxin resistant 1 mutants, which lack SMAP1, are more sensitive to MLN4924 than wild type and that SMAP1 is responsible for this hypersensitivity. This new evidence supports our previous speculation that SMAP1 acts in Cullin-RING ubiquitin E3 ligase regulated signaling processes via its interaction with components associated with the RUB modification system.     

MLN4924 is an efficient inhibitor of NEDD8 conjugation in plants

The conjugation of the ubiquitin-like modifier NEURAL PRECURSOR CELL-EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN8/RELATED TO UBIQUITIN1 (NEDD8/RUB1; neddylation) is best known as an important posttranslational modification of the cullin subunits of cullin-RING-type E3 ubiquitin ligases (CRLs). MLN4924 has recently been described as an inhibitor of NEDD8-ACTIVATING ENZYME1 (NAE1) in human. Here, we show that MLN4924 is also an effective and specific inhibitor of NAE1 enzymes from Arabidopsis (Arabidopsis thaliana) and other plant species. We found that MLN4924-treated wild-type seedlings have phenotypes that are highly similar to phenotypes of mutants with a partial defect in neddylation and that such neddylation-defective mutants are hypersensitive to MLN4924 treatment. We further found that MLN4924 efficiently blocks the neddylation of cullins in Arabidopsis and that MLN4924 thereby interferes with the degradation of CRL substrates and their downstream responses. MLN4924 treatments also induce characteristic phenotypes in tomato (Solanum lycopersicum), Cardamine hirsuta, and Brachypodium distachyon. Interestingly, MLN4924 also blocks the neddylation of a number of other NEDD8-modified proteins. In summary, we show that MLN4924 is a versatile and specific neddylation inhibitor that will be a useful tool to examine the role of NEDD8- and CRL-dependent processes in a wide range of plant species.     

A novel protein modification pathway related to the ubiquitin system.

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.     

The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis

Xie and colleagues previously isolated the Arabidopsis COI1 gene that is required for response to jasmonates (JAs), which regulate root growth, pollen fertility, wound healing, and defense against insects and pathogens. In this study, we demonstrate that COI1 associates physically with AtCUL1, AtRbx1, and either of the Arabidopsis Skp1-like proteins ASK1 or ASK2 to assemble ubiquitin-ligase complexes, which we have designated SCF(COI1). COI1(E22A), a single amino acid substitution in the F-box motif of COI1, abolishes the formation of the SCF(COI1) complexes and results in loss of the JA response. AtRbx1 double-stranded RNA-mediated genetic interference reduces AtRbx1 expression and affects JA-inducible gene expression. Furthermore, we show that the AtCUL1 component of SCF(COI1) complexes is modified in planta, where mutations in AXR1 decrease the abundance of the modified AtCUL1 of SCF(COI1) and lead to a reduction in JA response. Finally, we demonstrate that the axr1 and coi1 mutations display a synergistic genetic interaction in the double mutant. These results suggest that the COI1-mediated JA response is dependent on the SCF(COI1) complexes in Arabidopsis and that the AXR1-dependent modification of the AtCUL1 subunit of SCF(COI1) complexes is important for JA signaling.     

Related to ubiquitin 1 and 2 are redundant and essential and regulate vegetative growth, auxin signaling, and ethylene production in Arabidopsis

Related to Ubiquitin (RUB)/Nedd8 is a ubiquitin-like protein that covalently attaches to cullins, a subunit of the SCF (for Skp, Cdc53p/Cul1, and F-box protein) complex, an E3 ubiquitin ligase, and has been shown to be required for robust function of the complex. The effects of reducing protein levels for two Rub proteins, RUB1 and RUB2, were characterized in Arabidopsis thaliana. T-DNA insertional null lines homozygous at a single RUB-encoding locus were analyzed and found to have a wild-type phenotype. A double mutant was never recovered. More than one-quarter of the progeny from the self-fertilization of plants with a single functional RUB-encoding gene died as embryos at the two-cell stage. Outcrosses demonstrated reduced inheritance of the null allele from both the male and female parent. Hemigglutinin-tagged forms of RUB1 and RUB2 conjugate to the same cullin protein, CUL1, and produce the same conjugation pattern. To further understand the function of the RUB proteins, a construct designed to produce a double-stranded RUB1 mRNA was introduced into plants, and three lines with reduced levels of RUB1- and RUB2-encoding mRNA and RUB1/2 protein content were analyzed in detail. Mature plants were severely dwarfed, seedlings were insensitive to auxin in root assays, and dark-grown seedlings had a partial triple-response phenotype that was suppressed when seedlings were grown on ethylene perception or synthesis inhibitors. The dsrub lines produced threefold to fivefold more ethylene than the wild type. This study illustrates that RUB1 and RUB2 are genetically and biochemically redundant and demonstrates that RUB1/2 proteins are essential for early embryonic cell divisions and that they regulate diverse processes.     

Ubiquitin and Ubiquitin-like Modifiers in Plants

Posttranslational modifications of proteins by small polypeptides including ubiquitination, neddylation (related to ubiquitin (RUB) conjugation), and sumoylation are implicated in plant growth and development, and they regulate protein degradation, location, and interaction with other proteins. Ubiquitination mediates the selective degradation of proteins by the ubiquitin (Ub)/proteasome pathway. The ubiquitin-like protein RUB is conjugated to cullins, which are part of a ubiquitin E3 ligase complex that is involved in auxin hormonal signaling. Sumoylation, by contrast, is known for its involvement in guiding protein interactions related to abiotic and biotic stresses and in the regulation of flowering time. ATG8/ATG12-mediated autophagy influences degradation and recycling of cellular components. Other ubiquitin-like modifiers (ULPs) such as homology to Ub-1, ubiquitin-fold modifier 1, and membrane-anchored Ub-fold are also found in Arabidopsis. ULPs share similar three-dimensional structures and a conjugation system, including E1 activating enzymes, E2 conjugation enzymes, and E3 ligases, as well as proteases for deconjugation and recycling of the tags. However, each of the ULP posttranslational modifications possesses its own specific enzymes and modifies its specific targets selectively. This review discusses recent findings on ubiquitination and ubiquitin-like modifier processes and their roles in the posttranslational modification of proteins in Arabidopsis.     

Proteasome-dependent proteolysis has a critical role in fine-tuning the feedback inhibition of cytokinin signaling

The ubiquitin/26S proteasome-dependent proteolysis of response regulators is a critical element of many plant hormone signaling pathways. We have recently shown that cytokinin signaling requires the AXR1 component of the related to ubiquitin (RUB) protein modification pathway to promote the proteasome-dependent degradation of the cytokinin response inhibitor ARR5. Here, we show that ARR5 also accumulates in the 26S proteasome mutant rpn12a-1, and leads to a marked resistance to cytokinins. Collectively, these results suggest that proteasome-dependent proteolysis of feedback inhibitors such as ARR5 is essential for the maintenance of optimal responsivity and plasticity in cytokinin signaling.     

DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

The evolutionarily conserved 8-kD protein NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8) belongs to the family of ubiquitin-like modifiers. Like ubiquitin, NEDD8 is conjugated to and deconjugated from target proteins. Many targets and functions of ubiquitylation have been described; by contrast, few targets of NEDD8 have been identified. In plants as well as in non-plant organisms, the cullin subunits of cullin-RING E3 ligases are NEDD8 conjugates with a demonstrated functional role for the NEDD8 modification. The existence of other non-cullin NEDD8 targets has generally been questioned. NEDD8 is translated as a precursor protein and proteolytic processing exposes a C-terminal glycine required for NEDD8 conjugation. In animals and yeast, DENEDDYLASE1 (DEN1) processes NEDD8. Here, we show that mutants of a DEN1 homolog from Arabidopsis thaliana have no detectable defects in NEDD8 processing but do accumulate a broad range of NEDD8 conjugates; this provides direct evidence for the existence of non-cullin NEDD8 conjugates. We further identify AUXIN RESISTANT1 (AXR1), a subunit of the heterodimeric NEDD8 E1 activating enzyme, as a NEDD8-modified protein in den1 mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins.     

Light Regulates the RUBylation Levels of Individual Cullin Proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In plants, the small protein related to ubiquitin (RUB) modifies cullin (CUL) proteins in ubiquitin E3 ligases to allow for efficient transfer of ubiquitin to substrate proteins for degradation by the 26S proteasome. At the molecular level, the conjugation of RUB to individual CUL proteins is transient in nature, which aids in the stability of the cullins and adaptor proteins. Many changes in cellular processes occur within the plant upon exposure to light, including well-documented changes in the stability of individual proteins. However, overall activity of E3 ligases between dark- and light-grown seedlings has not been assessed in plants. In order to understand more about the activity of the protein degradation pathway, overall levels of RUB-modified CULs were measured in Arabidopsis thaliana seedlings growing in different light conditions. We found that light influenced the global levels of RUBylation on CULs, but not uniformly. Blue light had little effect on both Cul1 and Cul3 RUBylation levels. However, red light directed the increase in Cul3 RUBylation levels, but not Cul1. This red-light regulation of Cul3 was at least partially dependent on the activation of the phytochrome B signaling pathway. The results indicate that the RUBylation levels on individual CULs change in response to different light conditions, which enable plants to fine-tune their growth and development to the various light environments.     

A recessive mutation in the RUB1-conjugating enzyme, RCE1, reveals a requirement for RUB modification for control of ethylene biosynthesis and proper induction of basic chitinase and PDF1.2 in Arabidopsis

By screening etiolated Arabidopsis seedlings for mutants with aberrant ethylene-related phenotypes, we identified a mutant that displays features of the ethylene-mediated triple response even in the absence of ethylene. Further characterization showed that the phenotype observed for the dark-grown seedlings of this mutant is reversible by prevention of ethylene perception and is dependent on a modest increase in ethylene production correlated with an increase in 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) activity in the hypocotyl. Molecular characterization of leaves of the mutant revealed severely impaired induction of basic chitinase (chiB) and plant defensin (PDF)1.2 following treatment with jasmonic acid and/or ethylene. Positional cloning of the mutation resulted in identification of a 49-bp deletion in RCE1 (related to ubiquitin 1 (RUB1)-conjugating enzyme), which has been demonstrated to be responsible for covalent attachment of RUB1 to the SCF (Skpl Cdc 53 F-box) ubiquitin ligase complex to modify its activity. Our analyses with rce1-2 demonstrate a previously unknown requirement for RUB1 modification for regulation of ethylene biosynthesis and proper induction of defense-related genes in Arabidopsis.     

Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PP_i) exchange activity was inhibited by both AMP and PP_i. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5′-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.