Recombinant Arabidopsis thaliana RING-H2 finger protein ATL13 (ATL13)

What is ATL13 and how is it classified among RING finger proteins?

ATL13 (AT4G30400) is a member of the Arabidopsis Tóxicos en Levadura (ATL) family of RING-H2 type E3 ubiquitin ligases. It belongs to a plant-specific family of proteins named after the toxic phenotype exhibited by ATL2 (the first identified member) when conditionally expressed in Saccharomyces cerevisiae. The protein contains a RING-H2 finger domain, which is one of the two major types of RING domains found in Arabidopsis thaliana, the other being RING-HC . The RING-H2 domain is characterized by specific spacing between zinc ligands and the substitution of histidine for cysteine at specific positions in the zinc coordination pattern.

How does ATL13 function as an E3 ubiquitin ligase?

ATL13 functions as a RING-type E3 ubiquitin transferase in the ubiquitin-proteasome system. The RING-H2 finger domain mediates the interaction with E2 ubiquitin-conjugating enzymes, facilitating the transfer of ubiquitin to target proteins. This process marks these targets for degradation by the 26S proteasome. The specificity of substrate recognition is determined by regions outside the RING domain . ATL family proteins, including ATL13, are believed to play roles in various plant physiological processes through the regulated degradation of specific protein targets, although the specific substrates of ATL13 have not been fully characterized in the current literature.

What expression systems are effective for producing recombinant ATL13?

Based on available data, E. coli has been successfully used to express recombinant ATL13. Specifically, a full-length recombinant version (1-472aa) with an N-terminal His tag has been effectively produced in E. coli systems . This suggests that bacterial expression is suitable for obtaining functional protein for in vitro studies. When designing expression constructs, researchers should consider:

• Codon optimization for E. coli expression

• Selection of appropriate fusion tags (His tag has been demonstrated to work effectively)

• Optimization of induction conditions to maximize soluble protein yield

• Consideration of protein toxicity issues that might affect expression levels

What are the recommended storage conditions for maintaining ATL13 stability?

Recombinant ATL13 protein stability is maximized under the following conditions:

It is strongly advised to avoid repeated freeze-thaw cycles as they significantly compromise protein integrity and activity . Proper aliquoting upon initial reconstitution is critical for maintaining consistent results in downstream applications.

What reconstitution protocol is recommended for lyophilized recombinant ATL13?

For optimal reconstitution of lyophilized recombinant ATL13:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being commonly used)

• Prepare multiple small-volume aliquots to avoid repeated freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term storage or at 4°C for up to one week for active use

This protocol helps maintain the structural integrity and functional activity of the protein for subsequent experimental applications.

How can researchers effectively design in vitro ubiquitination assays using recombinant ATL13?

For in vitro ubiquitination assays using recombinant ATL13, researchers should consider the following methodological approach:

• Components required:

  • Purified recombinant ATL13 (RING-H2 E3 ligase)

  • E1 ubiquitin-activating enzyme

  • Appropriate E2 ubiquitin-conjugating enzyme (testing multiple E2s is advisable)

  • Ubiquitin (consider using tagged versions for easier detection)

  • ATP and ATP regeneration system

  • Putative substrate proteins (if known)

• Reaction conditions:

  • Buffer: Typically Tris-based (pH 7.5-8.0) with MgCl₂ and DTT

  • Temperature: 30-37°C

  • Duration: 1-2 hours or time course analysis

• Controls to include:

  • Negative control: Reaction without E3 ligase (ATL13)

  • Negative control: Reaction without ATP

  • Positive control: Well-characterized E3-substrate pair

• Detection methods:

  • Western blotting using anti-ubiquitin antibodies

  • If using tagged ubiquitin: detection via the tag (e.g., anti-His for His-tagged ubiquitin)

  • Using both anti-substrate and anti-ubiquitin antibodies to confirm ubiquitination

The key challenge in these assays is identifying the physiological substrates of ATL13, which may require additional approaches such as yeast two-hybrid screening or co-immunoprecipitation experiments to identify interacting proteins.

What approaches can be used to study the function of ATL13 in planta?

To investigate ATL13 function in Arabidopsis thaliana:

• Genetic approaches:

  • T-DNA insertion mutants analysis

  • CRISPR/Cas9-mediated gene editing

  • RNAi-mediated knockdown

  • Overexpression lines using the native promoter or constitutive promoters

• Protein localization studies:

  • GFP/YFP fusion proteins to determine subcellular localization

  • Immunolocalization using specific antibodies against ATL13

• Physiological and biochemical assays:

  • Phenotypic analysis under various stress conditions

  • Protein stability assays with and without proteasome inhibitors

  • Comparative proteomics between wild-type and atl13 mutants to identify accumulated proteins (potential substrates)

• Interaction studies:

  • Co-immunoprecipitation to identify interacting proteins

  • Bimolecular fluorescence complementation (BiFC) to confirm interactions in planta

  • Yeast two-hybrid screening to identify potential substrates and regulatory proteins

When designing these experiments, it's important to consider the potential functional redundancy with other ATL family members, which might necessitate the creation of multiple mutant lines.

How does the RING-H2 domain of ATL13 compare structurally to other ATL family members?

The RING-H2 domain of ATL13 shares the characteristic features of ATL family proteins but also has distinctive elements:

• Conserved features across ATL family:

  • The canonical RING-H2 pattern with specific spacing between zinc-coordinating residues

  • The central region encompassing the third to sixth residues involved in zinc binding is particularly informative for distinguishing RING finger protein subfamilies

• Distinguishing features of ATL13:

  • UniProt ID: Q940Q4

  • Specific residues in the RING domain that may influence E2 enzyme selection

  • Additional domains that work in conjunction with the RING-H2 domain

• Evolutionary considerations:

  • ATL13 belongs to a plant-specific family of RING-E3 ligases

  • Phylogenetic analysis based on the RING domain has been useful for cataloging and identifying conserved motifs in these proteins

Understanding these structural distinctions is crucial for predicting functional differences between ATL family members and for designing specific inhibitors or interactors.

What are common challenges in working with recombinant ATL13 and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant ATL13:

How can researchers verify the functional activity of purified recombinant ATL13?

To confirm that purified recombinant ATL13 retains its E3 ligase activity:

• Auto-ubiquitination assay:

  • Many RING E3 ligases can undergo auto-ubiquitination in vitro

  • This provides a straightforward functional assay even without known substrates

  • Reaction components: ATL13, E1, E2, ubiquitin, ATP

  • Detection via Western blot showing higher molecular weight species of ATL13

• Structural integrity verification:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Thermal shift assays to evaluate protein stability

  • Zinc content analysis to confirm proper metal coordination

• E2 binding assays:

  • Pull-down assays with different E2 enzymes

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic binding parameters

These approaches provide complementary information about the structural integrity and functional capacity of the recombinant protein.

What analytical techniques are most useful for studying ATL13 interactions with E2 enzymes and substrates?

Several analytical techniques are particularly valuable for characterizing interactions between ATL13 and its partners:

• For E2-E3 interactions:

  • Yeast two-hybrid (Y2H) screening with various E2 enzymes

  • Pull-down assays using tagged recombinant proteins

  • SPR or bio-layer interferometry (BLI) for real-time interaction kinetics

  • NMR spectroscopy for structural details of the interaction interface

• For substrate identification and characterization:

  • Protein microarrays to screen for ubiquitination targets

  • Tandem affinity purification coupled with mass spectrometry (TAP-MS)

  • Quantitative proteomics comparing wild-type and atl13 mutant plants

  • Proximity-dependent biotin identification (BioID) in planta

• For validating and characterizing specific interactions:

  • Mutagenesis of key residues to disrupt specific interactions

  • Co-crystal structure determination of ATL13 with partners

  • In vitro competition assays to assess binding specificity

  • Cellular co-localization studies to confirm physiological relevance

How might comparative studies between ATL13 and other ATL family members inform functional specialization?

Comparative studies between ATL13 and other ATL family members represent an important research direction:

• Phylogenetic analysis:

  • Comprehensive phylogenetic analysis of ATL13 in relation to other ATL proteins can reveal evolutionary relationships

  • The central region of the RING-H2 domain is particularly informative for classification

  • Identification of ATL13 orthologs across plant species can provide insights into conservation and functional importance

• Domain swapping experiments:

  • Exchanging domains between ATL13 and other ATL proteins to determine regions responsible for functional specificity

  • Creating chimeric proteins to test substrate specificity determinants

  • Systematically mutating residues that differ between ATL family members

• Expression and localization patterns:

  • Comparative analysis of expression patterns under various conditions

  • Comparison of subcellular localization to identify potential functional divergence

  • Co-expression analysis to identify functional networks

• Substrate specificity:

  • Comparing the substrate profiles of different ATL proteins

  • Determining the structural basis for substrate recognition differences

  • Investigating potential overlapping functions and redundancy

These comparative approaches could reveal how ATL13 has evolved specialized functions within the broader ATL family context.

What are the most promising approaches for identifying physiological substrates of ATL13?

Identifying the physiological substrates of ATL13 is a critical step in understanding its function:

• Global proteomics approaches:

  • Quantitative proteomics comparing wild-type and atl13 mutant plants

  • Ubiquitinome analysis to identify differentially ubiquitinated proteins

  • Protein stability profiling to identify proteins with altered half-lives

• Targeted interaction screens:

  • Yeast two-hybrid screens using ATL13 as bait

  • Protein microarrays to identify direct ubiquitination targets

  • Proximity-dependent labeling (BioID or TurboID) in plant cells

• Candidate-based approaches:

  • Testing proteins involved in pathways where ATL13 has been implicated

  • Investigating proteins that co-localize with ATL13

  • Examining proteins with expression patterns that inversely correlate with ATL13

• Validation strategies:

  • In vitro ubiquitination assays with candidate substrates

  • Co-immunoprecipitation to confirm interactions

  • Genetic interaction studies (e.g., epistasis analysis)

  • Analysis of candidate substrate levels in response to ATL13 overexpression or knockout

The combination of these approaches will likely be necessary to comprehensively identify and validate ATL13 substrates.

How can researchers effectively design experiments to elucidate ATL13 function in specific plant processes?

To effectively investigate ATL13 function in specific plant processes, researchers should consider a multi-layered experimental approach:

• Phenotypic characterization:

  • Generate and characterize atl13 knockout/knockdown lines

  • Create ATL13 overexpression lines

  • Analyze phenotypes under various conditions (developmental stages, stress treatments)

  • Look for subtle phenotypes that might be masked by redundancy

• Expression analysis:

  • Determine spatiotemporal expression patterns using promoter-reporter constructs

  • Analyze expression changes in response to various stimuli

  • Compare with expression patterns of other ATL family members

• Functional genomics:

  • RNA-seq analysis of atl13 mutants to identify affected pathways

  • ChIP-seq to identify potential transcriptional effects

  • Metabolomics to detect changes in metabolite profiles

• Protein function studies:

  • Create versions with mutations in key domains (RING domain, transmembrane domain)

  • Assess protein-protein interactions in planta

  • Determine the subcellular localization under various conditions

This comprehensive approach maximizes the likelihood of uncovering the specific biological processes in which ATL13 participates, even in the presence of potential functional redundancy with other E3 ligases.

What considerations are important when using recombinant ATL13 in structural studies?

For structural studies of recombinant ATL13, researchers should consider:

• Protein preparation considerations:

  • Expression of individual domains versus full-length protein

  • Selection of appropriate tags and cleavage sites

  • Protein homogeneity requirements (size-exclusion chromatography)

  • Buffer optimization to prevent aggregation and promote stability

• Structural technique selection:

  • X-ray crystallography: Requires high concentration and crystallization conditions

  • NMR spectroscopy: Suitable for individual domains, requires isotope labeling

  • Cryo-EM: Potentially useful for complexes with E2 enzymes or substrates

  • Small-angle X-ray scattering (SAXS): For low-resolution envelope information

• Potential complexes to study:

  • ATL13 RING domain with E2 enzymes

  • ATL13 in complex with substrate proteins

  • ATL13 transmembrane domain in membrane mimetics

• Functional validation:

  • Correlating structural features with biochemical activity

  • Structure-guided mutagenesis to validate functional hypotheses

  • Comparing structural features with other ATL family members

Careful consideration of these factors will enhance the likelihood of success in structural studies and provide valuable insights into ATL13 function.