Recombinant Arabidopsis thaliana E3 ubiquitin-protein ligase ATL42 (ATL42)

What is ATL42 and how is it classified within the E3 ubiquitin ligase family?

ATL42 (At4g28890) is a RING-H2 finger E3 ubiquitin ligase belonging to the ATL family in Arabidopsis thaliana. It is part of a large gene family comprising approximately 80 members in Arabidopsis and 121 in rice (Oryza sativa) . The ATL family is characterized by a highly conserved RING-H2 domain with a specific signature pattern: a highly conserved proline spaced one residue upstream from the third zinc ligand, and a conserved tryptophan spaced three residues downstream from the sixth zinc ligand . ATL42 is encoded by the At4g28890 gene and is also known by the synonyms F25O24.10 and RING-type E3 ubiquitin transferase ATL42 .

Methodologically, researchers can identify and classify ATL proteins through sequence alignment focusing on the conserved RING-H2 motif (C-X2-C-X(9-39)-C-X(1-3)-H-X(2-3)-C-X2-C-X(4-48)-C-X2-C) and the characteristic proline and tryptophan residues that distinguish the ATL family from other RING finger proteins .

What are the optimal conditions for expressing and purifying recombinant ATL42 protein?

Based on successful protocols with other ATL family members and the information from commercial sources , the following methodology is recommended:

• Expression System Selection: E. coli is the preferred expression system for ATL42. BL21(DE3) strain is commonly used for high yield protein expression .

• Construct Design:

  • Use the mature protein sequence (residues 19-432)

  • Add an N-terminal His-tag for purification

  • Clone into a pET or pGEX vector with an IPTG-inducible promoter

• Expression Conditions:

  • Grow transformed E. coli to OD600 of 0.6-0.8 at 37°C

  • Induce with 0.5-1.0 mM IPTG

  • Shift temperature to 18-20°C for overnight expression to increase solubility

• Purification Protocol:

  • Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, and protease inhibitors

  • Purify using Ni-NTA affinity chromatography

  • Elute with imidazole gradient (50-250 mM)

  • Further purify by size-exclusion chromatography if needed

• Storage Recommendations:

  • Store in Tris/PBS-based buffer with 6% trehalose at pH 8.0

  • Add 5-50% glycerol for long-term storage

  • Aliquot and store at -80°C to avoid repeated freeze-thaw cycles

For solubility issues, consider expressing only the RING domain or using fusion tags like MBP (maltose-binding protein) as successfully employed for other ATL family members .

How can I experimentally verify the E3 ubiquitin ligase activity of ATL42?

To verify the E3 ubiquitin ligase activity of ATL42, use an in vitro ubiquitination assay following protocols established for related proteins like ATL2 and ATL9 :

Materials required:

• Purified recombinant ATL42 (as GST or MBP fusion)

• Recombinant E1 (ubiquitin-activating enzyme, typically from yeast)

• Recombinant E2 (ubiquitin-conjugating enzyme, use AtUBC8 or human UbcH5b)

• Ubiquitin

• ATP and ATP regeneration system

• Reaction buffer (typically 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 2 mM DTT)

Experimental procedure:

• Set up the complete reaction containing E1, E2, ATL42, ubiquitin, and ATP

• Set up control reactions omitting each component individually

• Incubate at 30°C for 1-2 hours

• Analyze by SDS-PAGE followed by western blotting with anti-ubiquitin antibodies

Expected results:
The complete reaction should show multiple high-molecular-weight ubiquitinated bands, while control reactions missing any essential component should show no ubiquitination .

To identify critical residues for ligase activity, perform site-directed mutagenesis of conserved cysteine residues in the RING domain, as demonstrated for ATL2 where the Cys138Ala mutation abolished E3 ligase activity .

What methods can be used to identify potential substrate proteins of ATL42?

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

• Yeast Two-Hybrid Screening:

  • Create a bait construct with ATL42 (consider using only the substrate-binding domain to avoid auto-activation)

  • Screen against an Arabidopsis cDNA library

  • Validate interactions with co-immunoprecipitation assays in planta

• Proximity-Based Labeling:

  • Generate ATL42 fusion with BioID or TurboID

  • Express in Arabidopsis cells

  • Identify biotinylated proximal proteins by mass spectrometry

• Ubiquitination Assays with Candidate Substrates:

  • Select candidate substrates based on known functions of other ATL family members

  • Test ubiquitination of these candidates in vitro

  • Verify protein stability changes in vivo using ATL42 overexpression and knockout lines

• Proteomics Approach:

  • Compare ubiquitinated proteome in wild-type and atl42 mutant plants using tandem ubiquitin-binding entities (TUBEs) enrichment

  • Focus on proteins showing differential ubiquitination

• Genetic Interaction Screening:

  • Generate an ATL42 overexpression line

  • Cross with an Arabidopsis T-DNA insertion collection

  • Identify suppressors or enhancers of ATL42-related phenotypes

These methods should be used in combination for robust substrate identification, as established for other E3 ubiquitin ligases in plants .

What evidence suggests ATL42's involvement in plant defense mechanisms?

While direct evidence for ATL42's role in defense is limited in the provided search results, several lines of indirect evidence suggest its potential involvement:

• ATL Family Pattern: Multiple ATL family members, including ATL2 and ATL9, have been demonstrated to play essential roles in defense against fungal pathogens . For example, ATL2 is necessary for defense against the fungal pathogen A. brassicicola .

• Expression Patterns: Many ATL family members, including ATL2, are rapidly induced in response to elicitors, suggesting a role in early defense responses . A similar expression pattern for ATL42 would support its role in defense.

• Functional Conservation: Approximately 60% of rice ATLs are clustered with Arabidopsis ATLs, with many showing sequence similarities beyond the conserved features, suggesting conserved functions . This functional conservation within the family points to potential defense roles for ATL42.

• Regulation of Protein Stability: E3 ubiquitin ligases in plants often regulate the stability of defense-related proteins, controlling the turnover of signaling components, transcription factors, and receptors involved in pathogen recognition .

Methodological approaches to confirm ATL42's role in defense:

• Generate atl42 knockout mutants and test susceptibility to different pathogens

• Create ATL42-overexpressing plants and assess resistance phenotypes

• Analyze ATL42 expression patterns upon pathogen infection or PAMP treatment

• Identify ATL42 substrates related to defense signaling

• Perform epistasis analysis with known defense pathway components

How might ATL42 function in abiotic stress response pathways?

E3 ubiquitin ligases play crucial roles in abiotic stress responses by modulating protein stability in response to environmental changes. For ATL42:

• Potential Role in Hormone Signaling:

  • Many ATL family members are involved in hormone signaling pathways, especially those related to stress responses

  • ATL43, a family member, showed an ABA-insensitive phenotype when mutated, suggesting a role in ABA response

  • ATL42 might similarly regulate components of hormone signaling pathways related to drought, salinity, or temperature stress

• Protein Quality Control:

  • Under stress conditions, E3 ligases often target misfolded or damaged proteins for degradation

  • ATL42 may participate in this quality control mechanism during abiotic stress

• Experimental Approaches to Investigate:

  • Analyze ATL42 expression under different abiotic stresses (drought, salt, cold, heat)

  • Compare stress tolerance of wild-type vs. atl42 mutant plants

  • Identify changes in the ubiquitinated proteome under stress conditions in wild-type vs. atl42 mutants

  • Perform yeast two-hybrid or co-IP experiments to identify stress-specific interaction partners

• Quantitative Trait Locus (QTL) Analysis:

  • Utilize advanced intercross-recombinant inbred lines (AI-RILs) like those described for Arabidopsis

  • Map QTLs for stress tolerance traits and determine if ATL42 colocalizes with any identified QTLs

  • This approach has been effective for identifying genes involved in various stress responses

How can I determine the critical residues for ATL42's E3 ligase activity using site-directed mutagenesis?

Based on studies of related ATL proteins, a systematic approach to identify critical residues in ATL42 would include:

• Identification of Conserved Residues:

  • Align ATL42 with other characterized ATL proteins, particularly ATL2 where Cys138 was shown to be essential for activity

  • Focus on conserved cysteine and histidine residues in the RING-H2 domain that coordinate zinc ions

  • Also examine conserved residues surrounding the RING domain that might affect E2 interaction

• Site-Directed Mutagenesis Strategy:

  • Generate point mutations of conserved cysteines to alanine (e.g., C→A)

  • Create mutations of conserved histidines to alanine (H→A)

  • Target the conserved tryptophan characteristic of ATL family members

  • Consider conservative vs. non-conservative mutations to assess structural vs. functional roles

• Experimental Verification:

  • Express and purify wild-type and mutant proteins

  • Perform in vitro ubiquitination assays as described for ATL2

  • Compare activity levels between wild-type and mutant proteins

  • Conduct structural analysis (e.g., circular dichroism) to determine if mutations affect protein folding

• In Vivo Functional Complementation:

  • Transform atl42 mutant plants with constructs expressing mutated versions of ATL42

  • Assess whether mutant versions can complement the phenotype

  • This approach can determine if E3 ligase activity is essential for the biological function of ATL42

• E2 Binding Assays:

  • Perform pull-down assays between wild-type/mutant ATL42 and various E2 enzymes

  • Identify residues specifically involved in E2 recognition vs. catalytic activity

This methodology successfully identified Cys138 as critical for ATL2's ubiquitin ligase activity, with the C138A mutation completely abolishing E3 activity while maintaining protein stability .

What techniques can be used to investigate the subcellular localization and membrane integration of ATL42?

To determine the subcellular localization and membrane integration of ATL42, several complementary approaches can be employed:

• Fluorescent Protein Fusion and Confocal Microscopy:

  • Generate N- and C-terminal GFP fusions of ATL42

  • Express in Arabidopsis protoplasts or stable transgenic plants

  • Image using confocal microscopy

  • Co-localize with established organelle markers (ER, Golgi, plasma membrane)

• Biochemical Fractionation:

  • Isolate different subcellular fractions from plants expressing tagged ATL42

  • Analyze protein distribution by western blotting

  • This approach successfully showed ATL2 localization to the plasma membrane

• Membrane Integration Analysis:

  • Perform protease protection assays to determine topology

  • Use membrane extraction with various detergents and salts to assess the strength of membrane association

  • Apply carbonate extraction (pH 11.5) to distinguish peripheral from integral membrane proteins

• Transmembrane Domain Analysis:

  • Create targeted deletions or mutations of predicted transmembrane domains

  • Observe changes in localization patterns

  • Use domain swapping with known membrane proteins to verify function

• Immunogold Electron Microscopy:

  • Provide high-resolution confirmation of subcellular localization

  • Particularly useful for distinguishing between closely associated membranes (e.g., ER vs. Golgi)

Based on findings for other ATL family members, ATL42 is likely integrated into a membrane system, possibly the plasma membrane as shown for ATL2 through bioinformatics, confocal imaging, and cell fractionation analysis .

How can I design and interpret experiments to assess ATL42's role in specific signaling pathways?

Designing experiments to elucidate ATL42's role in signaling requires a systematic approach:

• Generate and Characterize Genetic Materials:

  • Create atl42 knockout mutants using T-DNA insertion or CRISPR-Cas9

  • Develop ATL42 overexpression lines under constitutive and inducible promoters

  • Establish complementation lines with wild-type and mutated versions of ATL42

  • Consider the challenge of functional redundancy with other ATL family members

• Phenotypic Analysis Under Different Conditions:

  • Assess growth, development, and stress responses of genetic materials

  • Apply specific signaling pathway activators/inhibitors

  • Conduct dose-response curves and time-course experiments

  • Record quantitative phenotypic data (growth rates, stress tolerance metrics)

• Transcriptomic Analysis:

  • Perform RNA-seq comparing wild-type, knockout, and overexpression lines

  • Analyze under basal and induced conditions

  • Use gene set enrichment analysis (GSEA) to identify affected pathways

  • Compare with transcriptomic signatures of known signaling pathway mutants

• Protein-Protein Interaction Network:

  • Conduct immunoprecipitation followed by mass spectrometry (IP-MS)

  • Perform yeast two-hybrid screening with ATL42 as bait

  • Validate interactions using techniques like BiFC or FRET

  • Map ATL42 into known signaling networks

• Substrate Identification and Validation:

  • Compare ubiquitinated proteomes between wild-type and atl42 mutants

  • Focus on proteins showing altered stability in ATL42 mutants

  • Conduct in vitro ubiquitination assays with candidate substrates

  • Perform genetic epistasis tests with substrate mutants

• Interpretation Framework:

  • Use a systems biology approach to integrate multiple data types

  • Construct network models of ATL42 function

  • Consider redundancy and compensatory mechanisms

  • Compare with known functions of other ATL family members

The success of this approach is demonstrated by studies of ATL family members like ATL2, which was found to be essential for defense against the fungal pathogen A. brassicicola .

How does ATL42's structure and function compare with other well-characterized ATL family members?

A comparative analysis of ATL42 with other ATL family members reveals important structural and functional insights:

Methodologically, the comparative approach reveals:

• Functional Diversity: Despite structural similarities, ATL family members appear specialized for different signaling pathways and stress responses.

• Localization Patterns: Different ATL proteins localize to different membrane systems, suggesting distinct substrate pools and functions.

• E3 Ligase Mechanism: The conserved RING-H2 domain functions similarly across the family, with critical cysteine residues required for activity.

• Target Specificity: The regions outside the RING domain likely confer substrate specificity and determine the biological processes regulated.

These comparisons suggest experimental approaches for ATL42 characterization, using successful strategies employed for other family members while accounting for potential unique features.

What bioinformatic approaches can help predict ATL42 function based on evolutionary conservation?

Several bioinformatic approaches can provide insights into ATL42 function:

• Phylogenetic Analysis:

  • Construct phylogenetic trees of ATL family members across plant species

  • Identify ATL42 orthologs and determine evolutionary distances

  • Cluster analysis to identify functionally related subgroups

  • This approach revealed that about 60% of rice ATLs cluster with Arabidopsis ATLs, suggesting conserved functions

• Motif and Domain Analysis:

  • Identify conserved motifs outside the RING-H2 domain

  • Analyze conservation patterns of specific amino acids

  • Predict functional sites using tools like MEME, PRINTS, or PROSITE

  • The ATL family is characterized by specific conserved residues, including a proline near the third zinc ligand and a tryptophan downstream of the sixth zinc ligand

• Co-expression Network Analysis:

  • Use publicly available transcriptomic data to identify genes co-expressed with ATL42

  • Perform Gene Ontology enrichment analysis on co-expressed genes

  • Map ATL42 into functional networks using tools like ATTED-II

• Structural Prediction and Modeling:

  • Generate 3D structural models of ATL42 using homology modeling

  • Compare with known structures of RING domains

  • Predict protein-protein interaction interfaces

  • Identify potential substrate binding regions

• Genomic Context Analysis:

  • Analyze genomic neighborhood conservation (synteny)

  • Identify conserved non-coding sequences that may regulate expression

  • The fact that 90% of ATL genes are intronless suggests they evolved as functional modules

• Integrative Approach:

  • Combine multiple bioinformatic predictions

  • Weight predictions based on confidence scores

  • Validate top predictions experimentally

These approaches have successfully predicted functions for other E3 ligases and can guide experimental design for ATL42 characterization.

What are the common challenges in working with recombinant E3 ubiquitin ligases like ATL42 and how can they be overcome?

Researchers working with E3 ubiquitin ligases face several challenges:

• Protein Solubility Issues:

  • Challenge: RING domain proteins often aggregate during expression

  • Solution: Express at lower temperatures (16-20°C), use solubility tags (MBP, SUMO), optimize buffer conditions with additives like arginine and glutamic acid

• Maintaining Structural Integrity:

  • Challenge: Zinc coordination in RING domains is sensitive to oxidation and pH

  • Solution: Include reducing agents (DTT, TCEP) in all buffers, avoid metal chelators, maintain pH 7.0-8.0, consider adding zinc to purification buffers

• Identifying Physiological Substrates:

  • Challenge: In vitro ubiquitination may not reflect in vivo specificity

  • Solution: Use proximity labeling approaches, develop quantitative ubiquitinome analysis, combine genetic and biochemical approaches

• Detecting E3 Ligase Activity:

  • Challenge: Background activity and distinguishing auto-ubiquitination from substrate ubiquitination

  • Solution: Include appropriate controls, use mutated versions lacking ligase activity, employ fluorescent ubiquitin for quantitative assays

• Functional Redundancy:

  • Challenge: Other ATL family members may compensate for ATL42 loss

  • Solution: Generate multiple knockout lines, use inducible dominant-negative approaches, consider tissue-specific phenotypes

• Storage Stability:

  • Challenge: Purified E3 ligases often lose activity during storage

  • Solution: Store in buffer with trehalose (6%) , add glycerol (5-50%), make single-use aliquots to avoid freeze-thaw cycles, flash-freeze in liquid nitrogen

These challenges can be addressed through careful experimental design and optimization, as demonstrated in successful studies of other ATL family proteins like ATL2 and ATL9 .

How can I design effective controls for experiments involving ATL42 E3 ligase activity?

Proper controls are essential for experiments with E3 ubiquitin ligases:

• In Vitro Ubiquitination Assays:

  • Negative Controls:

    • Omit E1 enzyme to confirm E1-dependent activation

    • Omit E2 enzyme to verify E2-dependent conjugation

    • Omit ATP to confirm energy requirement

    • Omit ATL42 to detect background activity

    • Use catalytically inactive ATL42 mutant (RING domain cysteine mutated)

  • Positive Controls:

    • Include a well-characterized E3 ligase with known activity

    • Use a known E3-substrate pair as a system control

• Substrate Identification Experiments:

  • Specificity Controls:

    • Compare binding/ubiquitination with unrelated proteins

    • Perform competition assays with potential substrates

    • Test substrate specificity across multiple conditions

  • Technical Controls:

    • Include input controls for all pull-down experiments

    • Use isotype control antibodies for immunoprecipitations

    • Validate all tagged constructs for functionality

• In Vivo Studies:

  • Genetic Controls:

    • Use multiple independent knockout or overexpression lines

    • Include empty vector transformants for overexpression studies

    • Complement knockout lines with wild-type and mutant versions

  • Phenotypic Controls:

    • Compare with mutants of related ATL genes

    • Include positive controls for each phenotypic assay

    • Test multiple environmental conditions

• Protein-Protein Interaction Studies:

  • Binding Specificity Controls:

    • Test interaction with structurally similar non-substrates

    • Perform domain mapping to identify specific interaction regions

    • Include known non-interacting proteins

The importance of comprehensive controls is demonstrated in the study of ATL2, where systematic omission of reaction components confirmed the specific E3 ligase activity of the protein .

This methodological approach ensures reliable and reproducible results when working with complex enzymatic systems like E3 ubiquitin ligases.

What emerging technologies could advance our understanding of ATL42's role in plant biology?

Several cutting-edge technologies show promise for investigating ATL42 function:

• CRISPR-Based Technologies:

  • CRISPRi/CRISPRa: For fine-tuned expression control of ATL42

  • Base editing: To introduce specific mutations without DNA breaks

  • Prime editing: For precise genetic modifications of ATL42

  • CRISPR screens: To identify genetic interactions with ATL42

• Advanced Imaging Techniques:

  • Super-resolution microscopy: To visualize ATL42 subcellular localization at nanometer resolution

  • Live-cell imaging with optogenetics: To control ATL42 activity with light and observe real-time effects

  • Correlative light and electron microscopy (CLEM): To connect functional observations with ultrastructural details

• Proteomics Innovations:

  • Targeted proteomics (PRM/MRM): For precise quantification of ATL42 and substrates

  • Crosslinking mass spectrometry (XL-MS): To map interaction interfaces

  • UbiSite and UbiScan: For comprehensive mapping of ubiquitination sites

  • Proximity-dependent biotin identification (BioID/TurboID): To identify ATL42-proximal proteins in living cells

• Single-Cell Technologies:

  • Single-cell RNA-seq: To understand cell-type-specific roles of ATL42

  • Single-cell proteomics: To measure protein-level changes at cellular resolution

  • Spatial transcriptomics: To map ATL42 expression patterns in intact tissues

• Structural Biology Approaches:

  • Cryo-EM: To determine structures of ATL42 complexes

  • Integrative structural biology: Combining multiple structural techniques

  • AlphaFold2 and structure prediction: To model ATL42 interactions with substrates and E2 enzymes

• Systems Biology Integration:

  • Multi-omics data integration: To place ATL42 in regulatory networks

  • Machine learning approaches: To predict ATL42 substrates and functions

  • Network modeling: To understand system-wide effects of ATL42 perturbation

These technologies could significantly accelerate our understanding of ATL42 function beyond what has been achieved for other ATL family members.

What are the most pressing unanswered questions about ATL42 that researchers should prioritize?

Based on the current state of knowledge, several key questions about ATL42 warrant investigation:

• Substrate Identification and Specificity:

  • What are the physiological substrates of ATL42?

  • How does ATL42 achieve substrate specificity?

  • Are substrates constitutively recognized or only under specific conditions?

• Physiological Function:

  • What is the primary biological role of ATL42 in Arabidopsis?

  • Does ATL42 function in plant immunity like other ATL family members?

  • What phenotypes are associated with ATL42 knockout or overexpression?

• Regulation Mechanisms:

  • How is ATL42 expression regulated at the transcriptional level?

  • Are there post-translational modifications that regulate ATL42 activity?

  • Which signaling pathways modulate ATL42 function?

• E2 Enzyme Partnerships:

  • Which E2 ubiquitin-conjugating enzymes preferentially work with ATL42?

  • Does ATL42 promote specific ubiquitin chain topologies?

  • How do E2-ATL42 interactions influence substrate selection?

• Structural Determinants of Function:

  • What is the three-dimensional structure of ATL42?

  • Which residues outside the RING domain contribute to function?

  • How does membrane association influence ATL42 activity?

• Evolutionary Context:

  • Why has the ATL family expanded so extensively in plants?

  • How has subfunctionalization occurred within the ATL family?

  • Are there fundamental differences between ATL42 orthologs across plant species?

• Integration in Plant Systems:

  • How does ATL42 function coordinate with other ubiquitin ligases?

  • Does ATL42 participate in specific developmental transitions?

  • How does ATL42 contribute to plant environmental responses?