Recombinant Arabidopsis thaliana RING-H2 finger protein ATL57 (ATL57)

What is the structural composition of ATL57?

ATL57, like other members of the ATL family, contains the characteristic RING-H2 domain that is essential for its E3 ubiquitin ligase activity. The RING-H2 domain features a precise arrangement of 8 zinc ligands with specific conserved amino acid residues . The protein likely contains three main structural regions: (1) the RING-H2 domain that binds to E2 ubiquitin-conjugating enzymes, (2) a hydrophobic region that potentially functions as a transmembrane domain, and (3) a GLD region (named after three conserved amino acids) whose function remains under investigation .

The hydrophobic region typically comprises 19 assorted sequence LOGOs, while the GLD is a highly conserved motif across the ATL family. Most ATL proteins harbor a single hydrophobic region, though some lineages possess two or three such regions .

How is ATL57 expressed in different tissues of Arabidopsis thaliana?

Based on expression patterns observed in other ATL family members such as ATL12, ATL57 is likely expressed across multiple plant tissues. For instance, histochemical staining of pATL12-GUS showed continuous expression in roots, leaves, stems, and flowers of Arabidopsis thaliana . Researchers interested in determining the specific expression pattern of ATL57 should consider generating transgenic plants expressing a promoter-reporter construct (such as pATL57-GUS) and performing histochemical staining across different developmental stages and tissues.

Expression analysis techniques should include:

• RT-qPCR analysis of different tissues

• Promoter-reporter fusion constructs for visualization

• RNA-seq data analysis across different developmental stages

What is the subcellular localization of ATL57?

While specific localization data for ATL57 has not been definitively established, many ATL family proteins localize to the plasma membrane. For example, subcellular co-localization of ATL12-GFP fusion protein with a plasma membrane-mcherry marker confirmed that ATL12 localizes to the plasma membrane .

To determine ATL57's subcellular localization, researchers should:

• Generate an ATL57-GFP fusion construct under a suitable promoter

• Express this construct in Arabidopsis or a heterologous system

• Perform confocal microscopy with appropriate subcellular markers

• Validate findings using biochemical fractionation methods

How is ATL57 related to other ATL family members?

ATL57 belongs to the larger ATL family of RING-H2 E3 ubiquitin ligases in Arabidopsis. This family is predicted to contain approximately 80 members, sharing common structural features including the RING-H2 domain, hydrophobic regions, and the GLD motif . Phylogenetic analysis would place ATL57 among other members based on sequence homology and domain architecture.

Researchers should conduct comparative sequence analysis to determine:

• The evolutionary relationships between ATL57 and other family members

• Potential functional redundancy with closely related ATLs

• Unique sequence features that might confer specific functions

What are the known functions of ATL57?

While specific functions of ATL57 remain to be fully characterized, insights can be drawn from other ATL family members. Many ATLs play crucial roles in plant defense responses. For example, ATL12 is involved in fungal defense, with atl12 mutants showing increased susceptibility to Golovinomyces cichoracearum infection .

ATL proteins often function in:

• Pathogen-associated molecular pattern (PAMP)-triggered immunity

• Hormone signaling pathways

• Stress responses

• Developmental processes

Researchers should investigate ATL57 function through knockout/knockdown mutants and overexpression lines, followed by phenotypic analysis under various stress conditions.

What are the optimal expression systems for recombinant ATL57 production?

For producing functional recombinant ATL57, several expression systems can be considered based on success with other RING-H2 proteins:

• Bacterial expression systems: E. coli BL21(DE3) strains with pET vectors are commonly used, but may require optimization for proper folding of the zinc finger domain. Consider including zinc in the culture medium (0.1-0.5 mM ZnSO₄) to promote proper folding of the RING-H2 domain.

• Insect cell expression systems: Baculovirus-infected insect cells (Sf9, Sf21, or High Five) often provide superior folding for plant proteins with complex domains compared to bacterial systems.

• Plant-based expression systems: Transient expression in Nicotiana benthamiana or stable expression in Arabidopsis cell cultures may preserve native post-translational modifications.

When expressing ATL57, researchers should consider:

• Using solubility tags (MBP, SUMO, or GST) to improve protein solubility

• Including protease inhibitors during purification to prevent degradation

• Optimizing zinc concentration in media and buffers to maintain RING-H2 domain integrity

• Testing different detergents if the hydrophobic domain creates solubility issues

How can ATL57 ubiquitination activity be effectively measured in vitro?

In vitro ubiquitination assays for ATL57 should be designed based on protocols used for other ATL family members. These typically require:

• Components:

  • Purified recombinant ATL57

  • E1 ubiquitin-activating enzyme

  • E2 ubiquitin-conjugating enzyme (preferably from the Ubc4/Ubc5 subfamily, which is known to work with ATL proteins)

  • Ubiquitin (unmodified or labeled)

  • ATP regeneration system

  • Appropriate buffer conditions

• Detection methods:

  • Western blotting with anti-ubiquitin antibodies

  • Using fluorescently labeled ubiquitin

  • Mass spectrometry to identify ubiquitination sites

• Controls:

  • RING-H2 domain mutant of ATL57 (negative control)

  • Omission of ATP (negative control)

  • Well-characterized E3 ligase (positive control)

What are the known E2 ubiquitin-conjugating enzymes that interact with ATL57?

Based on studies with other ATL family members, ATL57 likely interacts with E2 enzymes from the Ubc4/Ubc5 subfamily . The structural basis for E2-E3 recognition has been elucidated for EL5, a rice ATL protein, using NMR spectroscopy .

To identify specific E2 partners for ATL57, researchers should:

• Perform yeast two-hybrid or in vitro pull-down assays with different Arabidopsis E2 enzymes

• Test in vitro ubiquitination activity with various E2 enzymes

• Generate mutations in key residues of the RING-H2 domain to assess their impact on E2 binding

• Validate interactions using bimolecular fluorescence complementation in planta

Previous studies with ATL proteins have shown a good correlation between E3 activity and the degree of interaction between E2 enzymes and various RING domain mutants .

How does ATL57 expression change in response to different biotic stresses?

ATL family genes often show rapid and transient induction in response to pathogen-associated molecular patterns (PAMPs). For example, ATL2 is rapidly induced within 15-30 minutes following chitin treatment . ATL12 is similarly highly induced two hours after chitin treatment .

To analyze ATL57 expression in response to biotic stress:

• Time-course experiments: Monitor ATL57 transcript levels at multiple time points (15min, 30min, 1h, 2h, 6h, 24h) after treatment with:

  • Fungal PAMPs (chitin, β-glucans)

  • Bacterial PAMPs (flagellin, EF-Tu)

  • Pathogen infection (fungi, bacteria, oomycetes)

• Hormone treatments: Test response to defense hormones:

  • Salicylic acid (SA)

  • Jasmonic acid (JA)

  • Ethylene

• Protein synthesis inhibitor: Treat with cycloheximide to determine if ATL57 is a primary or secondary response gene, as ATL2 accumulation was found to be independent of de novo protein synthesis .

What are the known substrates of ATL57?

Identification of E3 ligase substrates remains challenging in plant systems. While specific substrates for ATL57 are not well-characterized, researchers can employ several approaches to identify potential targets:

• Yeast two-hybrid screening: Using ATL57 (with mutations in the RING domain to prevent auto-ubiquitination) as bait against an Arabidopsis cDNA library

• Co-immunoprecipitation coupled with mass spectrometry: Pull-down ATL57 complexes from plants expressing tagged ATL57 and identify associated proteins

• Proximity-dependent labeling: Use BioID or TurboID fusions with ATL57 to identify proteins in close proximity in vivo

• Proteomic analysis: Compare ubiquitinome profiles between wild-type and atl57 mutant plants to identify differentially ubiquitinated proteins

• Genetic suppressor screens: Identify mutations that suppress phenotypes of ATL57 overexpression or knockout lines

What are the recommended protocols for ATL57 protein purification?

Purification of functional recombinant ATL57 requires careful consideration of its RING-H2 domain integrity. A recommended protocol includes:

• Expression conditions:

  • Express in E. coli BL21(DE3) or Rosetta strain

  • Use low temperature induction (16-18°C) to improve folding

  • Supplement media with 0.1 mM ZnSO₄

  • Consider fusion tags that improve solubility (MBP, SUMO)

• Lysis and initial purification:

  • Lyse cells in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 0.1 mM ZnSO₄, 10% glycerol, protease inhibitors

  • For membrane-associated forms, include 0.5-1% non-ionic detergent (NP-40 or Triton X-100)

  • Clarify lysate by centrifugation (20,000 × g, 30 min)

• Chromatography steps:

  • Affinity chromatography using the fusion tag

  • Optional: Ion exchange chromatography

  • Size exclusion chromatography for final polishing

• Quality control:

  • SDS-PAGE and western blot analysis

  • Mass spectrometry to confirm identity

  • In vitro ubiquitination assay to confirm activity

How can I create and validate ATL57 knockout or overexpression lines?

To generate ATL57 transgenic lines:

• Knockout/knockdown strategies:

  • CRISPR-Cas9 targeting exons of ATL57 (preferably early exons or the RING-H2 domain)

  • T-DNA insertion mutants from available collections

  • RNA interference (RNAi) for knockdown studies

• Overexpression strategies:

  • Cloning ATL57 coding sequence under the CaMV 35S or native promoter

  • Generating fusion constructs with epitope tags (HA, FLAG) or fluorescent proteins (GFP)

  • Using inducible promoter systems (estradiol, dexamethasone) for controlled expression

• Validation methods:

  • Genotyping PCR for insertion/deletion confirmation

  • RT-qPCR for transcript level analysis

  • Western blotting for protein expression validation

  • Phenotypic characterization under normal and stress conditions

  • Complementation assays to confirm phenotype specificity

What methods are most effective for studying ATL57 protein-protein interactions?

Several complementary approaches should be used to study ATL57 interactions:

• In vivo techniques:

  • Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in plant cells

  • Co-immunoprecipitation from plant tissues expressing tagged ATL57

  • Förster Resonance Energy Transfer (FRET) for real-time interaction dynamics

  • Proximity labeling (BioID/TurboID) to capture transient interactions

• In vitro methods:

  • Pull-down assays with recombinant proteins

  • Surface Plasmon Resonance (SPR) for interaction kinetics

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Native gel electrophoresis for complex formation

• Computational approaches:

  • Molecular docking simulations

  • Protein-protein interaction network analysis

When studying specific interactions with E2 enzymes, researchers should focus on the RING-H2 domain and consider generating mutations in key residues, similar to studies performed with other ATL proteins .

How can I analyze ATL57 gene expression patterns in different conditions?

To comprehensively analyze ATL57 expression:

• Transcriptional analysis:

  • RT-qPCR for targeted expression analysis (with appropriate reference genes)

  • RNA-seq for genome-wide expression profiling

  • Promoter-reporter fusions (e.g., pATL57-GUS) for spatial expression patterns

• Temporal considerations:

  • Include early time points (15min, 30min, 1h) as many ATL genes show rapid and transient induction

  • Monitor expression over extended periods to capture expression dynamics

• Conditions to test:

  • Biotic stresses: fungal/bacterial PAMPs, pathogen infection

  • Abiotic stresses: drought, salt, cold, heat

  • Hormonal treatments: SA, JA, ethylene, abscisic acid

  • Developmental stages: seedling, vegetative, flowering, senescence

• Protein-level validation:

  • Western blot analysis with specific antibodies

  • Translational fusions with fluorescent proteins

What approaches can be used to identify potential substrates of ATL57?

Identifying E3 ligase substrates requires multiple complementary approaches:

• Interactome analysis:

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Yeast two-hybrid screening with substrate trapping mutants

  • Protein microarray screening with recombinant ATL57

• Ubiquitination target identification:

  • Ubiquitin remnant profiling comparing wild-type and atl57 mutants

  • In vitro ubiquitination assays with candidate substrates

  • Di-Gly antibody pulldown of ubiquitinated peptides followed by MS

• Genetic approaches:

  • Suppressor screens of atl57 phenotypes

  • Synthetic lethality/enhancement screens

  • Comparative transcriptome analysis to identify regulated pathways

• Informatics methods:

  • Analysis of co-expression networks

  • Protein interaction databases mining

  • Structural modeling of potential substrate binding sites

Why might recombinant ATL57 show low enzymatic activity in vitro?

Common issues affecting recombinant ATL57 activity and their solutions include:

• Improper folding of the RING-H2 domain:

  • Ensure sufficient zinc in expression media and purification buffers

  • Try different expression temperatures (16-25°C)

  • Consider refolding protocols with controlled zinc addition

• Incorrect E2 enzyme selection:

  • Test multiple E2 enzymes, particularly from the Ubc4/Ubc5 subfamily known to work with ATL proteins

  • Ensure the E2 enzyme is active by testing with a known E3 ligase

• Suboptimal reaction conditions:

  • Optimize buffer composition (pH, salt concentration)

  • Try different reducing agent concentrations (DTT vs. β-mercaptoethanol)

  • Adjust reaction temperature (room temperature vs. 30°C)

• Protein instability or aggregation:

  • Check protein by dynamic light scattering or size exclusion chromatography

  • Add stabilizing agents (glycerol, low concentrations of detergent)

  • Perform activity assays immediately after purification

How can I resolve inconsistent results between ATL57 in vitro and in vivo studies?

When in vitro and in vivo results for ATL57 function appear contradictory:

• Consider contextual factors in cellular environment:

  • Presence of cofactors or adaptor proteins missing in vitro

  • Post-translational modifications affecting activity

  • Subcellular compartmentalization that's lost in vitro

• Experimental design improvements:

  • Use cell extracts instead of purified components to bridge the gap

  • Develop semi-in vivo systems (e.g., permeabilized cells)

  • Recreate more physiological conditions in vitro (crowding agents, relevant pH)

• Technical validations:

  • Ensure protein functionality after tagging or purification

  • Validate antibody specificity

  • Use multiple independent methods to confirm results

• Genetic complementation:

  • Test if wild-type ATL57 rescues phenotypes in knockout lines

  • Test if active site mutants fail to complement

What are common pitfalls when studying ATL57 and how can they be avoided?

Common pitfalls in ATL research include:

• Functional redundancy: ATL family members may compensate for loss of ATL57 function.

  • Solution: Generate multiple mutants of closely related ATLs or use inducible dominant-negative approaches

• Transient expression changes: ATL genes often show rapid and transient induction .

  • Solution: Use detailed time courses with early time points and analyze transcript stability

• Protein instability: RING-H2 proteins may have short half-lives due to auto-ubiquitination.

  • Solution: Use proteasome inhibitors or create stabilized versions for interaction studies

• Non-specific interactions: E3 ligases may interact with multiple E2s and substrates.

  • Solution: Include appropriate controls and competition assays to validate specificity

• Artificial phenotypes: Overexpression may cause non-physiological effects.

  • Solution: Use complementation with native promoters and compare multiple independent lines

How should I analyze and interpret ATL57 protein interaction data?

When analyzing ATL57 interaction data:

• Establish meaningful controls:

  • Negative controls: unrelated proteins, RING domain mutants

  • Positive controls: known E2 partners or ATL family interactors

• Apply quantitative metrics:

  • Calculate affinity constants (Kd) for interactions when possible

  • Use statistical methods to distinguish specific from non-specific interactions

  • Consider stoichiometry of interactions

• Perform validation across methods:

  • Confirm key interactions using at least two independent techniques

  • Validate in both in vitro and in vivo systems

  • Use domain mapping to identify interaction interfaces

• Consider context:

  • Assess whether interactions are constitutive or condition-dependent

  • Determine if post-translational modifications affect interactions

  • Evaluate competition between different interactors

What are the best controls to include in ATL57 functional studies?

Robust ATL57 functional studies should include:

• Genetic controls:

  • Multiple independent transgenic lines

  • Complementation of knockout with wild-type gene

  • Active site mutants (RING-H2 domain mutations)

  • Related ATL family member knockouts for comparison

• Expression controls:

  • Verify transcript levels by RT-qPCR

  • Confirm protein expression and localization

  • Use appropriate housekeeping genes for normalization

• Phenotypic controls:

  • Wild-type plants grown under identical conditions

  • Known stress/defense response mutants as references

  • Treatment controls (mock, hormone/inhibitor vehicles)

• Biochemical controls:

  • Include enzymatically inactive versions in activity assays

  • Use structurally similar proteins to test binding specificity

  • Perform ATP-depleted controls in ubiquitination assays

• Technical controls:

  • Biological and technical replicates

  • Randomization of sample processing

  • Blinded phenotypic scoring when possible