Recombinant Arabidopsis thaliana RING-H2 finger protein ATL16 (ATL16)

What is Arabidopsis thaliana RING-H2 finger protein ATL16?

ATL16 (also known as At5g43420 or MWF20.13) is a member of the ATL family in Arabidopsis thaliana that encodes a RING-H2 finger domain protein. The protein functions as a RING-type E3 ubiquitin transferase, playing a crucial role in the ubiquitin/26S proteasome pathway that regulates protein degradation in plants . The full-length protein consists of 375 amino acids and contains characteristic domains including a transmembrane domain, a GLD motif following the transmembrane domain, and a RING-H2 finger domain that is essential for its E3 ligase activity .

How is ATL16 related to the broader ATL gene family?

ATL16 is one of 80 members of the ATL family identified in Arabidopsis thaliana. This family is part of a larger class of approximately 470 RING zinc-finger domain proteins that function as ubiquitin ligases . The ATL family is characterized by a highly conserved RING-H2 finger domain that is critical for ubiquitin ligase activity. Like about 90% of ATL genes, ATL16 is intronless, suggesting that the basic ATL protein structure evolved as a functional module. Comparative analysis with the 121 ATL members found in Oryza sativa (rice) shows that many Arabidopsis ATLs, including ATL16, have potential orthologous genes in rice with sequence similarities beyond the conserved ATL features .

How is recombinant ATL16 protein expressed and purified for research?

For expression of recombinant ATL16:

• Expression System: The full-length protein (amino acids 1-375) is typically expressed in E. coli with an N-terminal His tag .

• Vector Selection: A bacterial expression vector containing a strong promoter (such as T7) and an N-terminal His-tag sequence is recommended.

• Culture Conditions: Standard E. coli culture conditions with IPTG induction are effective for ATL16 expression.

• Purification Protocol:

  • Lyse cells in a Tris/PBS-based buffer

  • Perform affinity chromatography using Ni-NTA resin to capture the His-tagged protein

  • Wash extensively to remove contaminants

  • Elute with an imidazole gradient

  • Perform size exclusion chromatography if higher purity is required

  • Assess purity by SDS-PAGE (should be >90%)

  • Lyophilize the purified protein in Tris/PBS-based buffer with 6% Trehalose, pH 8.0

What are the optimal storage and handling conditions for recombinant ATL16?

For optimal stability and activity of recombinant ATL16:

• Storage Temperature: Store at -20°C/-80°C upon receipt.

• Aliquoting: Divide into small working aliquots to avoid repeated freeze-thaw cycles.

• Short-term Storage: Working aliquots can be stored at 4°C for up to one week.

• Reconstitution Protocol:

  • Briefly centrifuge the vial before opening

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

  • Add glycerol to a final concentration of 5-50% (recommended: 50%)

  • Aliquot for long-term storage at -20°C/-80°C

• Critical Precautions: Avoid repeated freeze-thaw cycles as they can significantly reduce protein activity

How can researchers design experiments to study ATL16's E3 ligase activity?

To investigate the E3 ubiquitin ligase activity of ATL16:

• In vitro Ubiquitination Assay:

  • Components needed: Purified recombinant ATL16, E1 enzyme, E2 enzyme (UBC), ubiquitin (preferably labeled), ATP, potential substrate proteins

  • Reaction buffer: Typically Tris-HCl (pH 7.5), MgCl₂, DTT, ATP

  • Controls: Reactions without E1, E2, ATL16, or ATP

  • Detection: Western blot analysis using anti-ubiquitin antibodies

• Substrate Identification:

  • Yeast two-hybrid screening to identify potential protein interactions

  • Co-immunoprecipitation with epitope-tagged ATL16

  • Proteomic analysis comparing ubiquitinated proteins in wild-type vs. ATL16 knockout plants

• Structure-Function Analysis:

  • Generate point mutations in the RING-H2 domain to disrupt zinc coordination

  • Compare ubiquitination activity of wild-type vs. mutant ATL16

  • Assess the role of other domains through truncation analysis

What approaches can be used to investigate ATL16's role in plant development and stress responses?

To elucidate ATL16's physiological functions:

• Genetic Approaches:

  • T-DNA insertion mutants or CRISPR/Cas9-generated knockout lines

  • Overexpression lines using constitutive (35S) or inducible promoters

  • Tissue-specific expression using appropriate promoters

  • Complementation studies with wild-type or mutated ATL16

• Expression Analysis:

  • qRT-PCR to determine tissue-specific expression patterns

  • Analysis under various stress conditions (drought, salinity, pathogen infection)

  • Reporter gene fusions (ATL16 promoter:GUS) to visualize expression patterns

  • Comparison with expression patterns of other ATL family members

• Phenotypic Analysis:

  • Morphological characterization at different developmental stages

  • Physiological responses to abiotic and biotic stresses

  • Comparison with phenotypes of other ATL family mutants

How can researchers address contradictions in experimental data when studying ATL16?

When confronted with contradictory data in ATL16 research:

• Structured Contradiction Analysis:

  • Identify the interdependent data items (α)

  • Enumerate the contradictory dependencies defined by domain experts (β)

  • Determine the minimal number of Boolean rules required to assess these contradictions (θ)

  • Apply notation (α, β, θ) to classify the contradiction pattern

• Methodological Approaches to Resolve Contradictions:

  • Validate key results using multiple experimental approaches

  • Ensure biological replicates are sufficient (n≥3)

  • Control for environmental variables that might affect plant phenotypes

  • Consider genetic background effects that might influence results

  • Test for gene redundancy among ATL family members

• Data Integration Strategies:

  • Implement structured classification of contradiction checks

  • Apply Boolean minimization techniques to handle complex interdependencies

  • Consider multidimensional relationships that might explain apparent contradictions

  • Document metadata thoroughly to support reproducibility

How does ATL16 compare with other members of the ATL family in terms of structure and function?

The ATL family in Arabidopsis comprises 80 members with varying degrees of sequence similarity and potentially different functions:

Key differences include substrate specificity, expression patterns, and responses to environmental cues. While ATL8 is mainly expressed in young siliques and appears essential for viability, ATL43 has been shown to be involved in ABA response pathways. These functional differences likely reflect evolutionary divergence despite structural conservation of the core RING-H2 domain .

What evolutionary insights can be gained from studying ATL16 in comparison to rice ATL homologs?

Comparative analysis of Arabidopsis ATL16 with rice ATL homologs reveals:

• Conservation Patterns:

  • Approximately 60% of rice ATLs cluster with Arabidopsis ATLs

  • Many show sequence similarities beyond the conserved ATL features

  • This suggests orthologous relationships and conserved functions

• Evolutionary Implications:

  • The high percentage of intronless genes (90%) suggests that ATL proteins evolved as functional modules

  • The expansion of the ATL family (80 in Arabidopsis vs. 121 in rice) indicates lineage-specific duplication events

  • Sequence divergence outside the RING-H2 domain likely reflects adaptation to different substrates and functions

• Functional Divergence Assessment:

  • Compare expression patterns between orthologous pairs

  • Analyze conservation of regulatory elements in promoter regions

  • Conduct complementation studies to test functional equivalence

  • Identify lineage-specific structural features that might confer novel functions

What are the common technical challenges in working with recombinant ATL16 and how can they be addressed?

Researchers often encounter several challenges when working with recombinant ATL16:

• Protein Solubility Issues:

  • Challenge: RING-H2 proteins can aggregate during expression

  • Solution: Express at lower temperatures (16-18°C), use solubility enhancing tags (MBP, SUMO), optimize buffer conditions with stabilizing agents (6% Trehalose)

• Maintaining Protein Activity:

  • Challenge: Loss of zinc coordination and structural integrity

  • Solution: Include zinc in purification buffers, add reducing agents (DTT or β-mercaptoethanol), avoid repeated freeze-thaw cycles

• Substrate Identification:

  • Challenge: Unknown physiological substrates complicates functional studies

  • Solution: Employ proteomics approaches comparing ubiquitination patterns in wild-type vs. ATL16 mutants, use proximity labeling techniques (BioID, APEX)

• Specificity in Ubiquitination Assays:

  • Challenge: Distinguishing specific from non-specific ubiquitination

  • Solution: Include appropriate controls (inactive RING mutants), verify with multiple E2 enzymes, confirm results with in vivo approaches

How can researchers optimize experimental design to study ATL16 in different plant tissues and developmental stages?

For comprehensive analysis of ATL16 across tissues and developmental stages:

• Expression System Selection:

  • Stable transformation: Use for consistent, heritable expression

  • Transient expression: Suitable for rapid testing in specific tissues

  • Inducible systems: Control expression timing for developmental studies

• Tissue-Specific Analysis Strategies:

  • Promoter:reporter constructs (ATL16pro:GUS or ATL16pro:GFP)

  • Tissue-specific promoters for targeted expression

  • Cell-type specific isolation techniques (FACS of GFP-marked cells)

  • Laser capture microdissection for precise tissue sampling

• Developmental Stage Considerations:

  • Synchronize plant growth for consistent developmental staging

  • Use multiple biological replicates at each developmental stage

  • Implement time-course experiments to capture dynamic changes

  • Compare results with expression data from public repositories

• Integration of Multiple Data Types:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Correlate expression patterns with phenotypic observations

  • Use network analysis to identify developmental stage-specific interactions