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.     

Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis

The AXR6 gene is required for auxin signaling in the Arabidopsis embryo and during postembryonic development. One of the effects of auxin is to stimulate degradation of the Aux/IAA auxin response proteins through the action of the ubiquitin protein ligase SCF(TIR1). Here we show that AXR6 encodes the SCF subunit CUL1. The axr6 mutations affect the ability of mutant CUL1 to assemble into stable SCF complexes resulting in reduced degradation of the SCF(TIR1) substrate AXR2/IAA7. In addition, we show that CUL1 is required for lateral organ initiation in the shoot apical meristem and the inflorescence meristem. These results indicate that the embryonic axr6 phenotype is related to a defect in SCF function and accumulation of Aux/IAA proteins such as BDL/IAA12. In addition, we show that CUL1 has a role in auxin response throughout the life cycle of the plant.     

Characterization of a novel temperature-sensitive allele of the CUL1/AXR6 subunit of SCF ubiquitin-ligases

Selective protein degradation by the ubiquitin-proteasome pathway has emerged as a key regulatory mechanism in a wide variety of cellular processes. The selective components of this pathway are the E3 ubiquitin-ligases which act downstream of the ubiquitin-activating and -conjugating enzymes to identify specific substrates for ubiquitinylation. SCF-type ubiquitin-ligases are the most abundant class of E3 enzymes in Arabidopsis. In a genetic screen for enhancers of the tir1-1 auxin response defect, we identified eta1/axr6-3, a recessive and temperature-sensitive mutation in the CUL1 core component of the SCF(TIR1) complex. The axr6-3 mutation interferes with Skp1 binding, thus preventing SCF complex assembly. axr6-3 displays a pleiotropic phenotype with defects in numerous SCF-regulated pathways including auxin signaling, jasmonate signaling, flower development, and photomorphogenesis. We used axr6-3 as a tool for identifying pathways likely to be regulated by SCF-mediated proteolysis and propose new roles for SCF regulation of the far-red light/phyA and sugar signaling pathways. The recessive inheritance and the temperature-sensitive nature of the pleiotropically acting axr6-3 mutation opens promising possibilities for the identification and investigation of SCF-regulated pathways in Arabidopsis.     

The eta7/csn3-3 Auxin Response Mutant of Arabidopsis Defines a Novel Function for the CSN3 Subunit of the COP9 Signalosome

The COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCF^TIR1/AFB ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit result in the pleiotropic cop/det/fus phenotype and cause seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In addition to enhancing tir1-1 mutant phenotypes, the csn3-3 mutation alone confers several phenotypes indicative of impaired auxin signaling including auxin resistant root growth and diminished auxin responsive gene expression. Unexpectedly however, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCF^TIR1-mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, we observe dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to another weak csn mutant, csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex is unaffected in csn3-3 plants. However, we detected a small CSN3-containing protein complex that is altered in csn3-3 plants. We hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a distinct protein complex that is required for proper auxin signaling.     

Targeting Proteins for Degradation by Arabidopsis COP1： Teamwork Is What Matters

Arsbidopsis COP1 （Constitutive Photomorphogenic 1） defines a key repressor of photomorphogenesis in darkness by acting as an E3 ubiquitin Iigase in the nucleus, and is responsible for the targeted degradation of a number of photomorphogenesis-promoting factors, including phyA, HY5, LAF1, and HFR1. Light activation of multiple classes of photoreceptors （including both phytochromes and cryptochromes） inactivates COP1 and reduces its nuclear abundance, allowing the accumulation of these positively acting light signaling intermediates to promote photomorphogenic development. Recent studies suggest that Arabidopsis COP1 teams up with a family of SPA proteins （SPA1-SPA4） to form the physiologically active COP1-SPA E3 ubiquitin ligase complexes. These COP1-SPA complexes play overlapping and distinct functions in regulating seedling photomorphogenesis under different light conditions and adult plant growth. Further, the COP1-SPA complexes act In concert at a biochemical level with the CDD （COP10, DET1, and DDB1） complex and COP9 signalosome （CSN） to orchestrate the repression of photomorphogenesis.     

The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses

The COP9 signalosome (CSN) is an evolutionarily conserved, nucleus-enriched multiprotein complex. CSN plays roles in photomorphogenesis, auxin response, and floral organ formation, possibly via the regulation of ubiquitin-proteasome-mediated protein degradation. COI1 encodes an F-box protein, which is a subunit of SCF(COI1) E3 ubiquitin ligase, and is required for jasmonate (JA) responses. Here, we demonstrate using coimmunoprecipitation and gel-filtration analyses that endogenous as well as epitope-tagged COI1 forms SCF(COI1) and associates directly with CSN in vivo. Like the coi1-1 mutant, CSN reduction-of-function plants exhibited a JA-insensitive root elongation phenotype and an absence of JA-induced-specific gene expression. Genome expression profile analyses indicated that JA-triggered genome expression is critically dependent on COI1 dosage. More importantly, most of the COI1-dependent JA-responsive genes also required CSN function, and CSN abundance was shown to be important for JA responses. Furthermore, we showed that both COI1 and CSN are essential for modulating the expression of genes in most cellular pathways responsive to JA. Thus, CSN and SCF(COI1) work together to control genome expression and promote JA responses.     

A role of Arabidopsis COP9 signalosome in multifaceted developmental processes revealed by the characterization of its subunit 3.

The COP9 signalosome is a highly conserved eight-subunit protein complex initially defined as a repressor of photomorphogenic development in Arabidopsis. It has recently been suggested that the COP9 signalosome directly interacts and regulates SCF type E3 ligases, implying a key role in ubiquitin-proteasome mediated protein degradation. We report that Arabidopsis FUS11 gene encodes the subunit 3 of the COP9 signalosome (CSN3). The fus11 mutant is defective in the COP9 signalosome and accumulates significant amount of multi-ubiquitinated proteins. The same mutant is specifically impaired in the 26S proteasome-mediated degradation of HY5 but not PHYA, indicating a selective involvement in protein degradation. Reduction-of-function transgenic lines of CSN3 produced through gene co-suppression also accumulate multi-ubiquitinated proteins and exhibit diverse developmental defects. This result substantiates a hypothesis that the COP9 signalosome is involved in multifaceted developmental processes through regulating proteasome-mediated protein degradation.     

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.     

Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8)

Arabidopsis COP1 is a negative regulator of photomorphogenesis, which targets HY5, a positive regulator of photomorphogenesis, for degradation via the proteasome pathway in the absence of light. COP1 and its interactive partner CIP8 both possess RING finger motifs, characteristic of some E3 ubiquitin ligases. Here we show that CIP8 promotes ubiquitin attachment to HY5 in E2-dependent fashion in vitro. CIP8 exhibits a strong interaction with the E2 enzyme AtUBC8 through its N-terminal domain. Phosphorylation of HY5 by casein kinase II requires the beta subunit 2, but does not affect HY5's susceptibility to ubiquitination. The RING domain of CIP8 is required but is not sufficient for ubiquitin ligase activity. Although the RING domain of CIP8 interacts with the RING domain of COP1, addition of recombinant COP1 fails to affect CIP8's ubiquitin ligase activity towards HY5 in vitro. However, recombinant COP1 can pull-down native CIP8 from the extract of dark-grown seedlings, but not from the extract of light-grown seedlings in a column-binding assay, implying a requirement for light-regulated modification in vivo. Our data suggest that CIP8 can form a minimal ubiquitin ligase in co-operation with the E2 enzyme AtUBC8. It is possible that the AtUBC8-CIP8 module might interact with COP1 in vivo, thereby participating in proteasome-mediated degradation of HY5.     

Characterization of the last subunit of the Arabidopsis COP9 signalosome: implications for the overall structure and origin of the complex

The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes. Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development. Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis. We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN. AtCSN2 is not only able to interact with the SCF(TIR1) subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCF(TIR1), but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions. Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution. Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests.     

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.     

The COP/DET/FUS proteins-regulators of eukaryotic growth and development

Eleven recessive mutant loci define the class of cop / det / fus mutants of Arabidopsis. The cop / det / fus mutants mimic the phenotype of light-grown seedlings when grown in the dark. At least four cop / det / fus mutants carry mutations in subunits of the COP9 signalosome, a multiprotein complex paralogous to the 'lid' subcomplex of the 26S proteasome. COP1, another COP/DET/FUS protein, is itself not a subunit of the COP9 signalosome. In the dark, COP1 accumulates in the nucleus where it is required for the degradation of the HY5 protein, a positive regulator of photomorphogenesis. In the light, COP1 is excluded from the nucleus and the constitutively nuclear HY5 protein can accumulate. Nuclear accumulation of COP1 and degradation of HY5 are impaired in the cop / det / fus mutants that carry mutations in subunits of the COP9 signalosome. Although the cellular function of the COP/DET/FUS proteins is not yet well understood, taken together the current findings suggest that the COP/DET/FUS proteins repress photomorphogenesis in the dark by mediating specific protein degradation.