Recombinant Arabidopsis thaliana RING-H2 finger protein ATL67 (ATL67)

What distinguishes RING-H2 domains from canonical RING domains?

The RING-H2 domain represents a subtle variation of the canonical RING finger domain, present in less than 10% of the RING fingers described in eukaryotes . The key difference lies in the substitution of the fifth zinc-coordinating cysteine residue with a histidine in the RING-H2 variant. This modification creates the C3HC3H structure instead of the C3HC4 structure found in canonical RING domains .

This variation is particularly significant in plants, where RING-H2 proteins like the ATL family appear to have evolved specialized functions. The conservation of this specific domain architecture in multiple plant gene families, but not widely in other eukaryotes, suggests that RING-H2 proteins fulfill plant-specific functions .

How can I obtain recombinant ATL67 protein for my experiments?

For successful production of recombinant ATL67, researchers should consider the following methodological approach:

• Vector selection: Choose expression vectors compatible with the transmembrane nature of ATL67. Consider vectors with solubility-enhancing tags (such as MBP or SUMO) to improve protein solubility.

• Expression system: E. coli BL21(DE3) or similar strains are commonly used for RING domain proteins, but due to the transmembrane domain, eukaryotic expression systems like insect cells might yield better results for full-length protein.

• Induction conditions: For E. coli systems, typical conditions include induction with 0.1-0.5 mM IPTG at OD600 of 0.6-0.8, followed by expression at lower temperatures (16-20°C) to enhance proper folding.

• Purification strategy: A two-step purification combining affinity chromatography and size exclusion chromatography is recommended. Buffer optimization is crucial, typically including:

  • 50 mM Tris-HCl, pH 7.5-8.0

  • 150-300 mM NaCl

  • 5-10% glycerol

  • 1 mM DTT or β-mercaptoethanol

  • 50 μM ZnCl2 (to stabilize the RING-H2 domain)

Commercially available recombinant ATL67 is typically supplied at concentrations of 50 μg in storage buffer containing Tris-based buffer with 50% glycerol .

What methods are most effective for studying ATL67 expression patterns?

To effectively study ATL67 expression patterns, researchers should employ a multi-tiered approach:

• Transcriptional analysis:

  • Quantitative RT-PCR offers high sensitivity for temporal expression patterns

  • RNA-seq provides genome-wide context for expression

  • Promoter-reporter fusions (ATL67pro:GUS or ATL67pro:GFP) enable tissue-specific localization studies

• Protein detection:

  • Western blotting with specific antibodies against ATL67 or epitope tags

  • Immunolocalization studies for cellular/subcellular distribution

• Response profiling: Since other ATL family members like ATL2 and ATL6 show early elicitor responses and sensitivity to cycloheximide , researchers should examine ATL67 expression under:

  • Treatment with elicitors (e.g., cellulase, chitin, flagellin)

  • Translation inhibitors (cycloheximide)

  • Hormonal treatments (JA, SA, ethylene, ABA)

  • Biotic and abiotic stress conditions

Studies of ATL2 and ATL6 have revealed rapid transcript accumulation after just 30 minutes of elicitor or cycloheximide treatment, with continued accumulation even after 120 minutes of incubation . Similar time-course experiments would be valuable for characterizing ATL67 responses.

How can I determine the subcellular localization of ATL67?

Given the presence of a putative transmembrane domain in ATL67, accurate subcellular localization determination is critical. A comprehensive approach would include:

• In silico prediction:

  • Use prediction tools like TMHMM, Phobius, or DeepLoc to identify potential transmembrane regions and targeting signals

  • Analyze the protein sequence for known localization motifs

• Fluorescent protein fusion approaches:

  • Generate both N- and C-terminal GFP/YFP fusions under native promoter control

  • Employ transient expression in Arabidopsis protoplasts or Nicotiana benthamiana leaves

  • Create stable transgenic Arabidopsis lines expressing ATL67-FP fusions

• Colocalization studies:

  • Use established organelle markers for membranes (PM, ER, Golgi, tonoplast)

  • Apply subcellular fractionation followed by Western blotting

  • Consider bimolecular fluorescence complementation (BiFC) if interaction partners are known

• Controls and validation:

  • Include analysis of truncated versions lacking the transmembrane domain

  • Perform protease protection assays for membrane topology

  • Use known ATL family members with established localization patterns as comparative controls

Most ATL family proteins localize to membrane structures, with many showing ER or plasma membrane localization patterns consistent with their transmembrane domains .

What is known about the E3 ubiquitin ligase activity of ATL67 and how can it be experimentally verified?

While specific data on ATL67's E3 ligase activity is limited in the provided search results, RING-H2 domains generally mediate E3 ubiquitin ligase activity. To verify this function for ATL67:

• In vitro ubiquitination assays:

  • Set up reactions containing purified components:

    • Recombinant ATL67 protein (E3)

    • E1 activating enzyme (e.g., UBA1)

    • E2 conjugating enzyme (test multiple E2s like UBC5, UBC8, UBC10)

    • Ubiquitin (preferably tagged, e.g., His-Ub or FLAG-Ub)

    • ATP and buffer components (Tris-HCl pH 7.5, MgCl2, DTT)

  • Run reactions with controls (minus E1, E2, E3, or ATP)

  • Detect ubiquitination by Western blot

• E2 specificity profiling:

  • Screen a panel of Arabidopsis E2s to identify the preferred E2 partner(s)

  • Quantify reaction kinetics with different E2s

• RING-H2 domain mutagenesis:

  • Generate point mutations in conserved zinc-coordinating residues

  • Compare activity of wild-type vs. mutant proteins

• In vivo ubiquitination:

  • Express epitope-tagged ATL67 and ubiquitin in plants

  • Immunoprecipitate potential substrates and probe for ubiquitination

  • Use proteasome inhibitors (MG132) to stabilize ubiquitinated proteins

How does ATL67 differ functionally from other ATL family members?

To elucidate the functional differences between ATL67 and other ATL family members, researchers should employ comparative analyses across multiple dimensions:

• Expression pattern comparison:
  Early response to elicitors has been demonstrated for ATL2 and ATL6, but not for ATL3, ATL4, and ATL5 . A systematic comparison of expression profiles across tissues, developmental stages, and in response to various stimuli would help position ATL67 within the family's functional spectrum.

• Phenotypic analysis of mutants:

  • Obtain and characterize T-DNA insertion or CRISPR-generated atl67 knockout lines

  • Create ATL67 overexpression lines

  • Design higher-order mutants with related ATL genes

  • Compare phenotypes under various stress conditions

• Substrate specificity:

  • Perform yeast two-hybrid or co-immunoprecipitation screens to identify interaction partners

  • Compare interactomes between ATL67 and other family members

  • Validate interactions with biochemical and in vivo approaches

• Structural comparison:

  • Analyze sequence conservation in regions outside the RING-H2 domain

  • Perform domain swaps between ATL proteins to identify regions responsible for specific functions

• Evolutionary analysis:

  • Compare selection pressures on different ATL genes

  • Analyze presence/absence of orthologs across plant species

Based on the available data, the ATL family appears to have undergone functional diversification, with some members specialized for early response to stimuli while others likely serve different functions .

What genome-wide approaches can be used to identify potential ATL67 substrates and interaction partners?

For comprehensive identification of ATL67 substrates and interactors, researchers should consider these advanced approaches:

• Proteomics-based methods:

  • Proximity-dependent biotin identification (BioID): Fuse ATL67 to BirA* biotin ligase to identify proximal proteins in vivo

  • Tandem affinity purification-mass spectrometry (TAP-MS): Use epitope-tagged ATL67 to pull down stable interaction partners

  • Ubiquitin remnant profiling: Compare ubiquitinomes of wild-type and atl67 mutant plants to identify differentially ubiquitinated proteins

• Protein-protein interaction screens:

  • Yeast two-hybrid screening: Use the RING-H2 domain or full-length ATL67 as bait

  • Split-ubiquitin assay: Particularly useful for membrane proteins like ATL67

  • Protein microarrays: Screen against Arabidopsis protein arrays

• Genetic approaches:

  • Suppressor/enhancer screens: Identify genetic modifiers of atl67 phenotypes

  • Synthetic lethality screening: Find genes with redundant or complementary functions

  • CRISPR-based screens: Deploy genome-wide CRISPR libraries in protoplasts

• Transcriptomics:

  • RNA-seq of atl67 mutants: Identify genes with altered expression

  • ChIP-seq of transcription factors: Determine if ATL67 regulates specific transcription factors

• Structural biology:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Map protein interaction interfaces

  • Cryo-EM or X-ray crystallography: Determine ATL67 structure in complex with substrates

What approaches can address the functional redundancy among ATL family members?

Functional redundancy is a common challenge when studying multigene families like the ATLs. The search results indicate at least 16 members of the ATL family in Arabidopsis , suggesting potential redundancy. To overcome this challenge:

• Higher-order mutant analysis:

  • Create double, triple, or higher-order mutants of phylogenetically-related ATL genes

  • Employ CRISPR/Cas9 multiplexing for simultaneous knockout of multiple ATL genes

  • Use artificial microRNAs to target conserved regions across multiple family members

• Tissue-specific and inducible approaches:

  • Generate tissue-specific knockdowns using promoter-specific CRISPR or RNAi

  • Develop inducible expression systems to overcome potential developmental defects

• Domain-based dominant negative strategies:

  • Express the RING-H2 domain alone to compete with endogenous ATL proteins

  • Create substrate-trapping versions with mutations that prevent substrate release

• Comparative analysis across family members:

  • Perform systematic phenotypic comparison of single and combinatorial mutants

  • Compare interactomes and ubiquitination targets across the family

  • Identify unique vs. shared regulatory elements in promoters

• Evolutionary approaches:

  • Study species with reduced genetic redundancy (e.g., basal land plants)

  • Compare with orthologs in other plant species with different family sizes

A systematic analysis of expression patterns, as performed for ATL2-6 , combined with phenotypic characterization of higher-order mutants, would provide valuable insights into shared and distinct functions within the ATL family.

How might ATL67's function relate to plant stress responses and immunity?

Given that some ATL family members (ATL2, ATL6) are early-response genes induced by elicitors , ATL67 may play roles in plant immunity and stress responses. To investigate this possibility:

• Expression profiling under biotic stress:

  • Challenge plants with pathogens of different lifestyles (bacteria, fungi, oomycetes, viruses)

  • Test with purified pathogen-associated molecular patterns (PAMPs) like flg22, chitin, or elf18

  • Analyze expression during different phases of immune responses

• Pathogen infection phenotyping:

  • Compare susceptibility of atl67 mutants vs. wild-type plants

  • Test multiple pathogen species to identify specific pathways affected

  • Measure standard immune outputs (ROS burst, callose deposition, defense gene expression)

• Exploration of signaling pathway placement:

  • Test genetic interactions with known immune regulators

  • Determine dependency on defense hormones (salicylic acid, jasmonic acid, ethylene)

  • Examine post-translational modifications of ATL67 during immune responses

• Potential immune-related substrates:

  • Screen for interactions with known pattern recognition receptors (PRRs)

  • Test involvement in regulating the stability of immune signaling components

  • Investigate role in degradation of negative regulators of immunity

Some research has indicated signals of selection in immune system-related genes that impart qualitative disease resistance to pathogens of bacterial and oomycete origins in Arabidopsis thaliana , making this a promising area for investigating ATL67 function.

What evolutionary patterns can be observed in the ATL family across plant species?

Understanding the evolutionary history of the ATL family provides context for ATL67's function. To explore this:

• Phylogenetic analysis:

  • Construct comprehensive phylogenies of ATL proteins across diverse plant species

  • Map conserved structural features and identify lineage-specific innovations

  • Determine orthologous relationships to guide functional predictions

• Comparative genomics:

  • Analyze synteny around ATL loci to identify genomic context conservation

  • Compare copy number variation across species with different evolutionary histories

  • Examine intron-exon structures for evidence of exon shuffling or domain acquisition

• Selection analysis:

  • Calculate Ka/Ks ratios to identify regions under positive or purifying selection

  • Perform site-specific selection analysis on the RING-H2 domain vs. other regions

  • Compare selection patterns between ATL67 and other family members

• Expression evolution:

  • Compare expression patterns of orthologous ATL genes across species

  • Analyze promoter evolution to identify conserved and divergent regulatory elements

• Functional conservation testing:

  • Perform cross-species complementation experiments

  • Test if orthologs from different species recognize the same substrates

Current evidence suggests that ATLs represent a plant-specific gene family , potentially emerging to fulfill specialized functions in plant development or stress responses. The conservation of the RING-H2 domain across multiple plant lineages points to its functional importance in plant-specific processes.

How can advanced structural biology approaches enhance our understanding of ATL67 function?

Structural insights can significantly advance functional understanding of ATL67:

• Structure determination approaches:

  • X-ray crystallography: Focus on the RING-H2 domain if full-length protein is challenging

  • Cryo-electron microscopy: Particularly useful for membrane-associated proteins

  • NMR spectroscopy: For dynamic regions and smaller domains

  • Integrative structural biology: Combine multiple methods with computational modeling

• Functional implications from structure:

  • Map substrate binding surfaces

  • Identify E2 interaction interfaces

  • Determine zinc coordination and its impact on domain stability

  • Understand membrane association mechanisms

• Structure-guided functional studies:

  • Design precise point mutations based on structural data

  • Create structure-based chimeric proteins to test domain functions

  • Develop structure-based inhibitors or activity modulators

• Dynamics and regulation:

  • Investigate conformational changes upon substrate binding

  • Examine potential post-translational modification sites

  • Study potential allosteric regulation mechanisms

• Computational approaches:

  • Molecular dynamics simulations to understand protein flexibility

  • Virtual screening to identify potential small molecule modulators

  • Protein-protein docking with E2 enzymes and potential substrates

Structural studies of ATL67 would contribute to broader understanding of how RING-H2 domains differ functionally from canonical RING domains and could reveal features unique to plant E3 ligases.

What technical challenges are common when working with membrane-associated E3 ligases like ATL67?

Membrane-associated E3 ligases present specific technical challenges that researchers should anticipate:

• Protein expression and purification issues:

  • Insolubility due to the transmembrane domain

  • Proper folding of the RING-H2 domain requiring zinc

  • Potential toxicity in expression hosts

  • Aggregation during purification

  Solution approaches:

  • Express soluble domains separately

  • Use detergents or amphipols for full-length protein

  • Consider nanodiscs or liposomes for native-like membrane environment

  • Test multiple expression systems (E. coli, insect cells, plant-based)

• Functional assay limitations:

  • Artificial in vitro conditions may not recapitulate membrane context

  • Difficulty in reconstituting physiological E2-E3 interactions

  • Identifying true substrates vs. promiscuous in vitro activity

  Solution approaches:

  • Develop membrane-based assay systems

  • Validate in vitro findings with in vivo approaches

  • Use proximity labeling in native cellular contexts

• Localization and trafficking considerations:

  • Determining precise subcellular localization

  • Understanding dynamic relocalization upon stimulation

  • Separating function from localization effects

  Solution approaches:

  • High-resolution microscopy techniques (STORM, PALM)

  • Live-cell imaging with minimal tags

  • Develop localization-specific activity assays

• Redundancy and specificity issues:

  • Overlapping functions with other ATL family members

  • Distinguishing specific vs. non-specific interactions

  • Substrate promiscuity in overexpression systems

  Solution approaches:

  • Higher-order mutants

  • Quantitative interaction proteomics

  • Domain swap experiments between family members

How can transcriptomics and proteomics be integrated to better understand ATL67 function?

A multi-omics approach provides more comprehensive insights into ATL67 function than any single method:

• Integrated experimental design:

  • Collect RNA-seq and proteomics data from the same biological samples

  • Include multiple timepoints to capture dynamic responses

  • Compare wild-type, atl67 mutant, and ATL67 overexpression lines

  • Include relevant treatments (elicitors, stresses) informed by ATL family function

• Correlation analysis:

  • Identify genes/proteins with correlated expression patterns

  • Build co-expression networks centered on ATL67

  • Compare transcriptional vs. post-translational regulation

• Pathway enrichment:

  • Perform integrated pathway analysis across transcriptome and proteome

  • Identify processes affected at both levels

  • Pinpoint pathways regulated primarily at protein stability level

• Ubiquitinome integration:

  • Combine standard proteomics with ubiquitin remnant profiling

  • Correlate changes in protein abundance with ubiquitination status

  • Identify direct vs. indirect effects of ATL67 activity

• Validation strategies:

  • Confirm key findings with targeted approaches (RT-qPCR, Western blotting)

  • Use time-course experiments to establish causality

  • Perform genetic validation of key targets

Integration of these datasets can distinguish between direct effects (e.g., ubiquitination and degradation of direct substrates) and downstream consequences (transcriptional responses to signaling changes), providing a systems-level understanding of ATL67 function.

How might understanding ATL67 function contribute to crop improvement strategies?

Research on ATL67 and related proteins has potential applications for crop improvement:

• Enhanced stress resistance:

  • If ATL67 regulates immunity or stress responses (as suggested by other ATL family members ), modulating its expression or activity could enhance crop resilience

  • Targeted breeding for optimal ATL67 alleles in crops with identified orthologs

  • Engineering of ATL67 or its substrates to optimize specific stress responses

• Developmental optimization:

  • Many E3 ligases regulate aspects of plant development

  • Understanding ATL67's role could allow fine-tuning of developmental processes

  • Potential applications in controlling flowering time, seed development, or architecture

• Pathway engineering:

  • Precise modification of ATL67 substrate specificity could allow targeted protein degradation

  • Creation of synthetic regulatory circuits incorporating ATL67-based components

  • Development of chemical tools to modulate ATL67 activity

• Cross-species applications:

  • Transfer of beneficial ATL67 alleles or engineered variants to crop species

  • Identification and improvement of crop orthologs based on ATL67 insights

  • Development of breeding markers based on ATL67 sequence or activity

• Climate adaptation strategies:

  • If ATL67 plays roles in stress responses, its optimization could contribute to climate resilience

  • Research suggests that environmental similarity between source populations and new environments is important for successful colonization in Arabidopsis , indicating potential value in studying environment-specific ATL variants

What emerging technologies might advance ATL67 research in the near future?

Several emerging technologies hold promise for deepening our understanding of ATL67:

• CRISPR technologies:

  • Base editors for precise modification of key residues without double-strand breaks

  • Prime editing for targeted nucleotide replacements with minimal off-target effects

  • CRISPR activation/repression systems for temporally controlled modulation

  • CRISPR screening approaches for genome-wide functional interaction mapping

• Advanced imaging:

  • Super-resolution microscopy for precise subcellular localization

  • Single-molecule tracking to follow ATL67 dynamics in living cells

  • Correlative light and electron microscopy for ultrastructural context

  • Expansion microscopy for enhanced resolution of protein complexes

• Protein engineering and analysis:

  • Directed evolution to identify ATL67 variants with enhanced or altered function

  • Proximity-dependent labeling techniques (TurboID, APEX) for in vivo interaction mapping

  • Non-canonical amino acid incorporation for site-specific protein modification

  • In-cell NMR for structural studies in native-like environments

• Single-cell approaches:

  • Single-cell transcriptomics to identify cell type-specific responses

  • Single-cell proteomics for protein-level heterogeneity analysis

  • Spatial transcriptomics to map expression patterns with tissue context

• Computational advances:

  • AlphaFold2 and related tools for improved structural prediction

  • Machine learning approaches for predicting E3-substrate pairs

  • Network analysis tools for integrating multi-omics datasets

  • Virtual screening for small molecule modulators of ATL67 activity

These technologies could overcome current limitations in studying membrane-associated E3 ligases and provide unprecedented insights into ATL67 function within complex cellular contexts.