Recombinant Arabidopsis thaliana RING-H2 finger protein ATL68 (ATL68)

What is ATL68 and what is its role in plant biological processes?

ATL68 (At3g61550) is a member of the Arabidopsis Tóxicos en Levadura (ATL) family of RING-H2 finger proteins that function as E3 ubiquitin ligases. The ATL family in Arabidopsis thaliana consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end . ATL68 is also referred to as a "RING/U-box superfamily protein" or "RING-type E3 ubiquitin transferase ATL68" .

The ATL family proteins participate in numerous biological processes including:

• Defense responses to pathogens

• Regulation of carbon/nitrogen response during post-germinative seedling growth

• Regulation of cell death during root development

• Endosperm development

• Transition to flowering under short day conditions

While the specific function of ATL68 has not been extensively characterized in the provided literature, as a member of the ATL family, it likely plays a role in protein ubiquitination and subsequent degradation through the 26S proteasome pathway.

How is the ATL gene family organized in Arabidopsis thaliana?

The ATL family is organized as follows:

• The Arabidopsis genome encodes 91 ATL members

• Some ATLs exist in clusters of tandem duplicated genes, suggesting evolution through gene duplication events

• The family can be divided into subgroups based on domain architecture and sequence conservation

• Members that share similar domain architecture often have similar expression patterns and may have related functions

For example, AthATL6 belongs to a subgroup of 11 ATLs that share common domain architecture and at least 5 of them are induced after cycloheximide treatment, suggesting they are early responsive genes .

How can recombinant ATL68 be expressed and purified for experimental studies?

Based on commercial production methods and general practices for RING-H2 proteins:

Expression Systems:

• E. coli expression systems are commonly used for recombinant RING finger proteins

• Yeast, baculovirus, or mammalian cell expression systems may be used for more complex post-translational modifications

Purification Strategy:

• Express the protein with an affinity tag (His-tag, GST, etc.)

• Lyse cells under conditions that maintain zinc coordination in the RING domain

• Purify using affinity chromatography

• Consider including zinc in buffers to maintain structural integrity of the RING-H2 domain

• Store in buffer containing glycerol at -20°C or -80°C to maintain stability

Storage Recommendations:

• Store at -20°C for regular use

• For long-term storage, store at -20°C or -80°C

• Avoid repeated freezing and thawing

• Working aliquots can be stored at 4°C for up to one week

What experimental approaches are effective for studying ATL68's E3 ligase activity?

Several approaches have been validated for studying ATL family proteins' E3 ligase activity:

In vitro Ubiquitination Assays:

• Combine purified recombinant ATL68 with:

  • Ubiquitin

  • E1 activating enzyme

  • Appropriate E2 conjugating enzyme (preferably from the Ubc4/Ubc5 subfamily)

  • ATP and buffer components

• Incubate and analyze ubiquitination products by Western blotting

E2 Selection:

• ATL family proteins primarily interact with members of the Ubc4/Ubc5 subfamily of E2 enzymes

• In Arabidopsis, there are 10 members of this subfamily that could potentially interact with ATL68

• Testing multiple E2 enzymes is recommended as specificity may vary

Substrate Identification:

• Yeast two-hybrid screening

• Co-immunoprecipitation coupled with mass spectrometry

• Protein microarrays

How can mutagenesis be used to investigate ATL68 function?

Strategic mutagenesis can provide insights into the structure-function relationships of ATL68:

RING-H2 Domain Mutations:

• Key amino acid residues for E2 binding can be identified through targeted mutations in the RING-H2 domain

• Studies on rice ATL (EL5) demonstrated a strong correlation between E3 activity and the degree of interaction between E2 enzymes and RING domain mutants

Mutation Design Strategy:

• Target conserved cysteine and histidine residues that coordinate zinc ions

• Mutate residues predicted to interact with E2 enzymes based on structural studies

• Create domain-swap chimeras with other ATL proteins to identify regions responsible for specificity

Phenotypic Analysis:

• Generate transgenic Arabidopsis lines with mutated versions of ATL68

• Analyze phenotypes under various stress conditions

• Compare with knockout mutants and overexpression lines

How does ATL68 contribute to plant responses to environmental stresses?

While specific information about ATL68's role in stress responses is limited in the provided literature, research on other ATL family members provides a framework for investigation:

Cold and Freezing Stress:

• Some ATL family members may be involved in cold stress responses

• A study on Arabidopsis freezing tolerance identified WRKY38 and LSD1 as genes contributing to drought and freezing tolerance through genotype-environment associations (GEA)

• Similar experimental approaches could be applied to study ATL68's potential role in cold stress adaptation

Experimental Design for Testing ATL68's Role in Stress:

• Monitor ATL68 expression under various stress conditions (drought, cold, salt, pathogen)

• Generate ATL68 knockout and overexpression lines

• Evaluate phenotypes under stress conditions

• Identify potential substrates that are differentially ubiquitinated during stress

Defense Responses:

• Several ATL proteins are involved in plant defense responses

• ATL2 expression is triggered after treatment with elicitors such as chitin

• Ectopic expression of ATL2 in eca mutants leads to expression of defense-related genes

• Similar approaches could be used to investigate ATL68's potential role in defense

What methods are most effective for identifying ATL68 substrates?

Identifying E3 ligase substrates is challenging but essential for understanding ATL68 function:

Integrated Approach for Substrate Identification:

• Protein Interaction Screening:

  • Yeast two-hybrid or split-ubiquitin assays

  • Protein microarray screening with recombinant ATL68

• Ubiquitinome Analysis:

  • Compare ubiquitinated proteins in wild-type vs. atl68 mutant plants using mass spectrometry

  • Enrich ubiquitinated proteins using tandem ubiquitin-binding entities (TUBEs)

• Dual-Layered Biological Network Analysis:

  • Construct protein-protein interaction networks combined with metabolic networks

  • Integrate expression data from ATL68 knockout/overexpression plants

  • This approach has been successful for studying resistance gene analogs in plants

• Validation Experiments:

  • In vitro ubiquitination assays with candidate substrates

  • Co-immunoprecipitation under native conditions

  • Cell-free degradation assays to confirm substrate regulation

How does ATL68 compare to other ATL family members in terms of structure and function?

Comparative analysis can provide insights into the specialization of ATL68 within the family:

Structural Comparison:

• ATL68 contains the characteristic RING-H2 domain and hydrophobic domain

• Sequence alignments with other ATL proteins can identify conserved and variable regions

• Phylogenetic analysis can place ATL68 within specific subgroups of the ATL family

Functional Diversity in the ATL Family:

• Different ATL proteins may target distinct substrates

• Expression patterns vary among ATL members

  • Some are constitutively expressed

  • Others are induced by specific stimuli (e.g., AthATL2 is induced by chitin)

• Not all ATLs exhibit the same phenotype when expressed in yeast (only a few, like ATL2 and ATL63, show toxicity)

Experimental Approach for Comparative Studies:

• Perform phylogenetic analysis of ATL family using RING-H2 domain sequences

• Compare expression patterns across different tissues and conditions

• Conduct complementation studies to test functional redundancy

• Analyze substrate specificity differences using in vitro assays

What role might ATL68 play in the circadian clock regulation in Arabidopsis?

While specific information about ATL68's role in circadian regulation is not provided in the search results, E3 ubiquitin ligases are known to play crucial roles in circadian clock regulation:

Circadian Clock Components in Arabidopsis:

• The circadian clock in Arabidopsis comprises multiple feedback loops involving genes such as TOC1, GI, LHY, and CCA1

• E3 ubiquitin ligases regulate the stability of clock components through targeted degradation

• The three-loop model provides a framework for understanding how additional components like ATL68 might function

Experimental Approach to Investigate ATL68's Role in Circadian Regulation:

• Monitor ATL68 expression over circadian cycles

• Analyze circadian phenotypes in atl68 mutants (period length, phase, amplitude)

• Test interactions between ATL68 and known clock components

• Identify whether clock proteins are substrates for ATL68-mediated ubiquitination

How can CRISPR/Cas9 genome editing be utilized to study ATL68 function?

CRISPR/Cas9 technology offers powerful approaches for studying ATL68:

Gene Knockout Strategies:

• Design sgRNAs targeting ATL68 coding regions

• Generate complete knockout lines to assess loss-of-function phenotypes

• Create tissue-specific or inducible knockouts using appropriate promoters

Domain-Specific Editing:

• Introduce specific mutations in the RING-H2 domain to disrupt E3 ligase activity while maintaining protein structure

• Modify the hydrophobic domain to alter subcellular localization

• Edit putative substrate recognition regions to alter specificity

Multiplex Editing:

• Target ATL68 alongside related ATL genes to address functional redundancy

• Edit both ATL68 and potential substrate genes to validate interactions

Promoter Editing:

• Modify the ATL68 promoter to alter expression patterns

• Introduce reporter genes to monitor expression under different conditions

How can quantitative genetics approaches be used to study ATL68's role in natural variation and adaptation?

ATL68 may contribute to natural variation in stress responses and adaptation:

Genome-Environment Association (GEA) Studies:

• GEA approaches have successfully identified genes involved in local adaptation to environmental conditions

• A recent study validated genes identified through GEA for drought adaptation in Arabidopsis

• Similar approaches could reveal whether ATL68 variants are associated with specific environmental conditions

Experimental Design for Natural Variation Studies:

• Analyze ATL68 sequence variation across Arabidopsis accessions

• Test for associations between ATL68 variants and environmental variables

• Conduct reciprocal transplant experiments with accessions carrying different ATL68 alleles

• Create near-isogenic lines differing only in ATL68 alleles to test fitness effects

Trade-off Analysis:

• Recent research shows trade-offs between freezing tolerance and reproductive fitness in Arabidopsis

• Investigate whether ATL68 variants contribute to this trade-off

• Measure fitness parameters in controlled environments and field conditions