Recombinant Arabidopsis thaliana E3 ubiquitin-protein ligase RING1 (ATL55)

What is ATL55/RING1 and what is its role in Arabidopsis thaliana?

ATL55 (also known as RING1, At5g10380) is a member of the Arabidopsis Tóxicos en Levadura (ATL) family, a group of plant-specific RING-type ubiquitin ligases. ATL55 functions as an E3 ubiquitin ligase that mediates the transfer of ubiquitin to specific target substrates, thereby regulating protein degradation through the ubiquitin-proteasome system .

The ATL family is characterized by a RING-H2 type zinc finger domain that is essential for E3 ligase activity, and ATL55 contains this conserved domain . Like other members of this family, ATL55 plays roles in plant adaptation to environmental stresses, particularly in immune responses . In particular, ATL55/RING1 has been implicated in plant immunity and stress response pathways .

Where is ATL55 protein localized in plant cells?

Based on studies of other ATL family members, ATL55 is likely localized to membrane structures within the plant cell. Most ATL proteins contain one or two N-terminal transmembrane-like hydrophobic regions that anchor them to cellular membranes .

For example, ATL2, another member of the ATL family, was shown to be predominantly localized to the plasma membrane through fractionation experiments and confocal microscopy of GFP fusion proteins . Similarly, ATL8 was found to localize to membrane-bound compartments, with signals at the periphery of cells and in dot-like structures in the cytosol that may represent endosomal compartments .

Given the structural similarities between ATL55 and other ATL family members, it is highly likely that ATL55 is also membrane-associated, possibly at the plasma membrane or in endosomal compartments.

What expression systems are optimal for recombinant ATL55 production?

• Expression construct design: The N-terminal hydrophobic transmembrane region often inhibits sufficient protein expression in E. coli. It's advisable to delete this region and express only from residue 71 to the C-terminus, as has been done successfully with other ATL proteins like ATL8 .

• Fusion tags: Using a fusion tag such as MBP (maltose-binding protein) or His-tag can improve solubility and facilitate purification. According to the product information, commercially available recombinant ATL55 is often produced with an N-terminal His-tag .

• Expression conditions: Expression in E. coli BL21 Star (DE3) strain has been reported for other ATL proteins and likely works for ATL55 as well .

• Protein form: The recombinant protein is typically supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE .

For storage, it's recommended to store the protein at -20°C/-80°C upon receipt, aliquoting as necessary to avoid repeated freeze-thaw cycles. Working aliquots can be stored at 4°C for up to one week .

How can I assess the E3 ligase activity of recombinant ATL55 in vitro?

To assess the E3 ligase activity of recombinant ATL55 in vitro, you can perform an ubiquitination assay. Based on methods used for other ATL family proteins, the following protocol is recommended:

• Components needed:

  • Purified recombinant ATL55 protein

  • E1 (ubiquitin-activating enzyme)

  • E2 (ubiquitin-conjugating enzyme, typically from the Ubc4/Ubc5 subfamily for ATL proteins)

  • Ubiquitin

  • ATP

  • Reaction buffer

• Reaction setup:

  • Incubate the components together at appropriate temperatures (typically 30°C) for varying time periods (0, 30, and 120 minutes)

  • Set up a control reaction using a mutant version of ATL55 where a conserved cysteine in the RING domain (likely C123 based on homology to ATL8) is replaced with serine

• Analysis:

  • Analyze the reactions by SDS-PAGE followed by western blotting with an anti-ubiquitin antibody

  • Active E3 ligase will produce a heterogeneous collection of higher molecular weight bands representing ubiquitinated proteins, while the mutant version should show no such activity

As a note, for ATL family proteins, the ubiquitin ligase activity has been shown to rely on members of the Ubc4/Ubc5 subfamily of E2 conjugases .

What are the critical residues in ATL55 that affect its E3 ligase activity?

Based on studies of other ATL family proteins, several critical residues in ATL55 are likely important for its E3 ligase activity:

• Conserved cysteines and histidines in the RING-H2 domain: The RING-H2 finger coordinates two zinc ions through a specific pattern of cysteine and histidine residues. For ATL proteins, the third cysteine in the RING motif (equivalent to C138 in ATL2 or C123 in ATL8) plays a crucial role in E3 ligase activity .

• Key residues for E2 interaction: Studies on EL5, a rice ATL protein, have identified key amino acid residues in the RING-H2 finger domain that are critical for binding to E2 conjugating enzymes. These residues showed good correlation between E3 activity and the degree of interaction with E2 enzymes .

• GLD motif: The highly conserved 12-16 amino acid motif that often begins with glycine, leucine, and aspartic acid residues (GLD motif) may also be important for the function of ATL55, though its specific role in E3 ligase activity is less clear .

To experimentally determine the critical residues, site-directed mutagenesis can be performed, followed by in vitro ubiquitination assays to assess the impact on E3 ligase activity. Typically, mutations of the conserved cysteines or histidines in the RING domain to serine or alanine abolish E3 ligase activity .

How is ATL55/RING1 gene expression regulated in Arabidopsis?

While specific information on ATL55/RING1 expression regulation is limited in the provided search results, insights can be drawn from studies of other ATL family members:

• Response to stress conditions: Many ATL genes, including ATL2 and ATL8, show increased expression in response to various stress conditions. For example, ATL2 expression is significantly upregulated following chitin treatment, indicating a role in pathogen defense responses .

• Tissue-specific expression: Some ATL genes show tissue-specific expression patterns. For instance, ATL5 is highly expressed in the embryos of seeds .

• Developmental regulation: ATL genes may be regulated during different developmental stages. For example, ATL5 expression is induced by accelerated aging in seeds .

To determine the specific expression pattern of ATL55/RING1, techniques such as quantitative RT-PCR, promoter-GUS fusion assays, or analysis of publicly available microarray or RNA-seq data can be used. Additionally, treatments with different stress conditions (e.g., pathogen elicitors, abiotic stresses) can help identify factors that induce ATL55 expression.

What are potential substrates of ATL55 and how can they be identified?

Identifying substrates of E3 ubiquitin ligases like ATL55 is challenging but several approaches can be employed:

• Yeast two-hybrid (Y2H) screening: This has been successful in identifying interacting proteins for other ATL family members. For example, a Y2H screen identified ABT1 as an interacting protein of ATL5 .

• Co-immunoprecipitation (Co-IP): This technique can confirm protein-protein interactions identified through Y2H or identify new interacting partners. For ATL5 and ABT1, the interaction was confirmed using Co-IP analysis .

• Bimolecular fluorescence complementation (BiFC): This method visualizes protein interactions in living cells and has been used to confirm interactions for other ATL proteins .

• In vitro and in vivo ubiquitination assays: These assays can determine if a potential substrate is ubiquitinated by the E3 ligase. For ATL5, both in vitro and in vivo assays showed that it mediates the polyubiquitination and degradation of ABT1 .

• Comparative proteomics: Comparing the proteome of wild-type plants with ATL55 knockout or overexpression lines can identify proteins whose abundance is affected by ATL55 activity.

Based on studies of other ATL family members, potential substrates might include:

• Transcription factors

• Signaling proteins involved in stress responses

• Proteins involved in plant immunity

• Proteins involved in hormone signaling pathways

For example, ATL5 targets ABT1 (ACTIVATOR OF BASAL TRANSCRIPTION 1) for degradation, which affects seed longevity in Arabidopsis .

How does ATL55 compare structurally and functionally to other ATL family members?

The ATL family in Arabidopsis consists of 91 members, all characterized by similar domain architecture . Here's how ATL55 compares to other family members:

Structural Comparisons:

• Domain architecture: Like other ATL proteins, ATL55 contains an N-terminal transmembrane domain, a GLD motif, and a RING-H2 finger domain .

• Phylogenetic relationships: ATL proteins have been classified into groups based on phylogenetic analysis of their RING domains. This classification can provide insights into the evolutionary relationships between ATL55 and other family members .

• Size and sequence: ATL55 consists of 301 amino acids, which is within the typical range for ATL proteins. Sequence conservation is highest in the RING-H2 domain and GLD motif, while the C-terminal regions show more divergence .

Functional Comparisons:

• Subcellular localization: Most ATL proteins, including likely ATL55, localize to membrane structures, though the specific membrane may vary (plasma membrane, endosomal compartments) .

• E3 ligase activity: All characterized ATL proteins function as E3 ubiquitin ligases, with the RING-H2 domain being essential for this activity .

• Biological roles:

  • ATL5: Positively regulates seed longevity by mediating the degradation of ABT1

  • ATL2: Plays a positive role in defense response against fungal pathogens

  • ATL8: Involved in sugar starvation response

  • ATL12: Involved in pattern-triggered immunity

The specific biological role of ATL55/RING1 appears to be related to plant immunity based on limited information in the search results , but more detailed studies would be needed to fully elucidate its function.

How can I design experiments to study ATL55's role in specific stress responses?

To investigate ATL55's role in specific stress responses, consider the following experimental approach:

• Genetic materials preparation:

  • Obtain T-DNA insertion mutants of ATL55 from repositories like the Arabidopsis Biological Resource Center (ABRC)

  • Generate ATL55-overexpressing lines under a constitutive promoter (e.g., CaMV35S)

  • Create transgenic lines expressing ATL55 with mutations in key residues (e.g., C123S mutation in the RING domain) to disrupt E3 ligase activity

• Expression analysis:

  • Perform RT-qPCR analysis to determine how ATL55 expression changes under various stress conditions (e.g., pathogen infection, abiotic stresses, hormone treatments)

  • Create promoter-GUS fusion constructs to visualize tissue-specific expression patterns

• Phenotypic analysis:

  • Challenge wild-type, atl55 mutant, and ATL55-overexpressing plants with various stresses:

    • Pathogen infection (e.g., fungal pathogens like Alternaria brassicicola)

    • Abiotic stresses (e.g., drought, salinity, temperature extremes)

    • Hormone treatments (e.g., jasmonic acid, salicylic acid, abscisic acid)

  • Assess phenotypic differences such as:

    • Disease resistance (lesion size, pathogen growth)

    • Growth parameters under stress

    • Physiological responses (e.g., reactive oxygen species production, callose deposition)

• Biochemical analysis:

  • Perform in vivo ubiquitination assays to determine if ATL55's E3 ligase activity changes under stress conditions

  • Investigate protein stability and turnover rates of ATL55 and its substrates under different conditions using cycloheximide chase assays

• Identification of molecular pathways:

  • Conduct RNA-seq analysis to identify genes differentially expressed between wild-type and atl55 mutant plants under stress conditions

  • Create double mutants with genes involved in known stress response pathways to identify genetic interactions

This comprehensive approach will help elucidate the specific role of ATL55 in plant stress responses and the molecular mechanisms involved.

What experimental design would be appropriate for identifying the role of ATL55 in seed longevity?

Given that ATL5, another member of the ATL family, positively regulates seed longevity by mediating the degradation of ABT1 , investigating ATL55's potential role in seed longevity would be valuable. Here's an experimental design approach:

1. Genetic Materials Preparation:

• Obtain homozygous T-DNA insertion mutants of ATL55

• Generate ATL55-overexpressing lines

• Create complementation lines (expressing ATL55 in atl55 mutant background)

• Develop transgenic lines expressing a catalytically inactive version of ATL55 (with mutations in key RING domain residues)

2. Seed Longevity Assessment:

3. Molecular Analysis:

• Analyze ATL55 expression during seed development, maturation, and aging using RT-qPCR

• Examine changes in potential substrates and associated proteins during seed aging

• Perform proteomic analysis to identify differentially accumulated proteins in atl55 mutants compared to wild-type during seed aging

4. Biochemical Characterization:

• Test if ATL55 expression is induced by seed aging conditions, similar to ATL5

• Investigate if ATL55 mediates the degradation of specific proteins during seed aging

• Assess oxidative damage markers and antioxidant enzyme activities in different genotypes

5. Potential Substrate Identification:

• Conduct Y2H screening to identify ATL55-interacting proteins

• Confirm interactions using BiFC and Co-IP analyses

• Test if identified interacting proteins are ubiquitinated by ATL55 in vitro and in vivo

• Examine if the stability of these proteins is affected in atl55 mutants during seed aging

This comprehensive experimental design would help determine if ATL55 plays a role in seed longevity and elucidate the underlying molecular mechanisms.

How can I investigate the auto-regulation of ATL55's stability and activity?

Auto-ubiquitination is a common regulatory feature of RING E3 ligases that affects their stability and activity . To investigate ATL55 auto-regulation, consider the following approaches:

• In vitro auto-ubiquitination assay:

  • Set up a standard ubiquitination reaction with E1, E2, ATP, and ubiquitin, but without adding a substrate

  • Include wild-type ATL55 and a catalytically inactive mutant (e.g., with a C123S mutation)

  • Analyze by western blotting using anti-ATL55 antibodies to detect auto-ubiquitination

  • The wild-type protein should show higher molecular weight bands indicative of auto-ubiquitination, while the mutant should not

• Protein stability analysis:

  • Perform cycloheximide (CHX) chase assays in plant cells expressing ATL55-HA and ATL55(C123S)-HA

  • Compare protein degradation rates between wild-type and mutant proteins

  • Based on studies with ATL2, the catalytically inactive mutant would likely show delayed degradation compared to the wild-type protein

• Proteasome inhibition experiments:

  • Treat plants expressing ATL55 with the proteasome inhibitor MG132

  • Analyze ATL55 protein levels with and without inhibitor treatment

  • If ATL55 is regulated by auto-ubiquitination, MG132 treatment should lead to increased protein levels

• Deubiquitinating enzyme interactions:

  • Investigate interactions between ATL55 and deubiquitinating enzymes (DUBs)

  • Based on studies in yeast, proteins like UBP15 (ortholog of human HAUSP/USP7) might protect ATL55 from auto-ubiquitination

  • Test if co-expression of specific DUBs affects ATL55 stability and activity

• Phosphorylation and other post-translational modifications:

  • Investigate if ATL55 undergoes phosphorylation or other modifications that might affect its auto-ubiquitination activity

  • Use phosphatase treatments and phospho-mimetic/phospho-dead mutations to assess the impact on ATL55 stability and activity

Understanding ATL55 auto-regulation will provide insights into how its activity is controlled in response to different cellular signals and environmental conditions.

What approaches can be used to study the evolutionary conservation and diversification of ATL55 across plant species?

To investigate the evolutionary conservation and diversification of ATL55 across plant species, consider the following approaches:

• Phylogenetic analysis:

  • Identify ATL55 orthologs in different plant species using BLAST searches against genomic databases

  • Align sequences and construct phylogenetic trees to determine evolutionary relationships

  • Analyze the distribution of ATL55 orthologs across plant lineages (e.g., monocots, dicots, non-flowering plants)

  • Previous studies have identified ATL family members in various plant species, with 91 in Arabidopsis, 119 in rice, and 82 in tomato

• Domain architecture comparison:

  • Analyze the conservation of key domains (transmembrane domain, GLD motif, RING-H2 domain)

  • Generate position-specific hidden Markov model (pHMM) LOGOs to visualize conserved motifs

  • Previous studies generated 75 pHMM LOGOs from 1815 ATLs to understand domain architecture

• Synteny and gene duplication analysis:

  • Examine the genomic context of ATL55 orthologs in different species

  • Identify syntenic regions to understand chromosomal rearrangements

  • Analyze tandem duplications, which appear to be a mechanism for species-specific expansion of ATL genes

• Selection pressure analysis:

  • Calculate Ka/Ks ratios (non-synonymous to synonymous substitution rates) to identify regions under positive or purifying selection

  • Compare selection patterns across different plant lineages and ATL subfamilies

• Functional conservation testing:

  • Perform complementation experiments by expressing ATL55 orthologs from different species in Arabidopsis atl55 mutants

  • Assess if orthologs can restore wild-type phenotypes

  • Compare biochemical properties (e.g., E3 ligase activity, substrate specificity) of ATL55 orthologs

• Expression pattern comparison:

  • Analyze expression data from different plant species to determine if ATL55 orthologs share similar expression patterns

  • Compare responses to stresses, developmental cues, and hormonal treatments