Recombinant Arabidopsis thaliana RING-H2 finger protein ATL32 (ATL32)

What is ATL32 and what is its relationship to other ATL proteins?

ATL32 (At4g40070) is a RING-H2 finger protein belonging to the ATL (Arabidopsis Toxicos en Levadura) family in Arabidopsis thaliana. It is part of a multigene family that includes other members like ATL2, ATL3, ATL4, ATL5, and ATL6 . This family is characterized by a specific variant of the RING zinc finger domain known as RING-H2, which is involved in protein-protein interactions and potentially in ubiquitin-mediated protein degradation . The ATL family appears to be plant-specific, with evidence suggesting conservation across multiple plant species including broccoli, pea, bean, tobacco, potato, tomato, rice, and maize .

What genetic information is available for ATL32?

ATL32 is encoded by the At4g40070 gene located on chromosome 4 of Arabidopsis thaliana. It is also known by the synonyms T5J17.240, RING-H2 finger protein ATL32, and RING-type E3 ubiquitin transferase ATL32 . The gene is a single-copy gene, though Southern blot analysis has shown some discrete regions of homology with other genes in the Arabidopsis genome .

How is ATL32 expression regulated in response to stress?

While the specific regulation of ATL32 isn't detailed in the provided search results, the regulation pattern of related ATL family members can provide insights. ATL family genes like ATL2 and ATL6 show rapid transcript accumulation in response to elicitors and cycloheximide treatment, suggesting their involvement in early stress responses .

The ATL family members may be part of plant stress response pathways that involve transcriptional reprogramming. In Arabidopsis, stress responses often involve regulatory modules like the ANAC044/ANAC085-mediated signaling pathway that perceives distinct stress signals and leads to cell cycle arrest . Though ATL32's specific role in these pathways isn't established in the provided search results, its structural similarity to other ATL proteins suggests potential involvement in stress-responsive transcriptional networks.

What evidence suggests ATL32 functions as an E3 ubiquitin ligase?

The primary evidence for ATL32's function as an E3 ubiquitin ligase comes from its domain structure:

• The presence of a RING-H2 finger domain, which is a characteristic feature of many E3 ubiquitin ligases

• Its classification as "RING-type E3 ubiquitin transferase ATL32" in database annotations

• Structural similarity to other ATL family members that have been characterized as E3 ligases

What expression systems are suitable for producing recombinant ATL32?

Based on the available information, E. coli has been successfully used to express recombinant full-length ATL32 protein (amino acids 29-323) with an N-terminal His-tag . The protein can be expressed as the mature form, excluding the first 28 amino acids which may include a signal peptide or other regulatory region.

For researchers considering expression systems, the following table summarizes key considerations:

The choice of expression system should depend on the specific research questions and whether post-translational modifications are critical for the planned experiments.

What are the optimal storage and handling conditions for recombinant ATL32?

According to the product information, recombinant ATL32 protein requires specific storage and handling conditions to maintain stability and activity :

• Storage temperature: -20°C to -80°C for long-term storage

• Physical form: Lyophilized powder as supplied

• Reconstitution: Using deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Storage buffer: Tris/PBS-based buffer containing 6% Trehalose, pH 8.0

• For long-term storage after reconstitution: Add glycerol to a final concentration of 5-50% (recommended 50%) and store in aliquots at -20°C/-80°C

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

• Avoid repeated freeze-thaw cycles as this may affect protein stability and activity

Prior to opening, it is recommended to briefly centrifuge the vial to bring contents to the bottom .

What methods can be used to assess the activity of recombinant ATL32?

To assess the functional activity of recombinant ATL32 as a putative E3 ubiquitin ligase, researchers could employ several methods:

• In vitro ubiquitination assays: Combining recombinant ATL32 with E1 activating enzyme, E2 conjugating enzyme, ubiquitin, ATP, and potential substrate proteins to detect ubiquitin transfer

• E2 binding assays: Using techniques such as pull-down assays or surface plasmon resonance to detect interaction between ATL32 and various E2 enzymes

• Zinc binding analysis: Confirming the proper folding and metal coordination of the RING-H2 domain through spectroscopic methods

• Substrate identification: Using yeast two-hybrid screens, co-immunoprecipitation, or protein arrays to identify potential target proteins

• Auto-ubiquitination assays: Testing whether ATL32 can facilitate its own ubiquitination, which is common for many RING E3 ligases

These methods would need to be optimized specifically for ATL32, as the search results do not provide specific protocols for this protein.

How can ATL32 be used to study stress response pathways in plants?

Researchers can use recombinant ATL32 protein and genetic approaches to study stress response pathways in plants through several advanced experimental designs:

• Identification of ATL32 substrates during stress responses: Using techniques such as proteomics combined with ubiquitination site mapping to identify proteins targeted by ATL32 under different stress conditions

• Analysis of ATL32 expression patterns: Developing ATL32 promoter-reporter constructs to monitor spatiotemporal expression patterns in response to various stresses

• Genetic manipulation: Creating ATL32 knockout/knockdown lines or overexpression lines to assess phenotypic effects on stress tolerance

• Protein interaction networks: Determining how ATL32 integrates into broader stress response networks, potentially interacting with signaling modules like the ANAC044/ANAC085 pathway that regulates cell cycle arrest during stress

• Comparative analysis with other ATL family members: Investigating functional redundancy or specialization among ATL family proteins in stress responses

For example, analyzing whether ATL32 participates in DNA damage response pathways similar to the ANAC044/ANAC085-Rep-MYB module that represses G2/M-specific genes during genotoxic and heat stress would provide valuable insights into its potential role in stress-induced cell cycle regulation.

What approaches can be used to identify the specific substrates of ATL32?

Identifying the specific substrates of E3 ubiquitin ligases like ATL32 is challenging but critical for understanding their biological functions. Several complementary approaches can be employed:

• Affinity purification coupled with mass spectrometry (AP-MS): Using tagged ATL32 to capture interacting proteins, followed by mass spectrometry identification

• Ubiquitination proteomics: Comparing the ubiquitinome (all ubiquitinated proteins) between wild-type plants and ATL32 mutants to identify differential ubiquitination

• Yeast two-hybrid screening: Systematic screening for proteins that physically interact with ATL32, particularly its non-RING domains

• Proximity-based labeling: Using techniques like BioID or TurboID fused to ATL32 to identify proteins in close proximity in vivo

• Genetic suppressor screens: Identifying mutations that suppress ATL32 overexpression or knockout phenotypes

• Domain-based prediction: Analyzing potential recognition motifs in the non-RING regions of ATL32 to predict classes of substrate proteins

• In vitro ubiquitination assays: Testing candidate substrates in reconstituted ubiquitination reactions with purified components

A combination of these approaches would provide the most comprehensive identification of ATL32 substrates.

How does ATL32 compare structurally and functionally to other members of the ATL family?

The ATL family in Arabidopsis comprises at least 16 members sharing several structural features. Based on the search results, we can infer the following comparisons:

Structural comparisons:

• All ATL family members possess a RING-H2 zinc finger domain with a conserved pattern of cysteine and histidine residues

• Most have a putative transmembrane domain, often located at the N-terminal end

• They contain additional regions of homology beyond the RING-H2 domain

Functional comparisons:

• ATL2 has been characterized as an early response gene with transcript levels increasing rapidly after elicitor treatment

• Some ATL members (ATL2, ATL6) show increased transcript accumulation after cycloheximide treatment, while others (ATL3, ATL4, ATL5) do not

• The family appears to be plant-specific, suggesting specialized functions in plant biology

A comprehensive comparative analysis of all ATL family members would require experimental data on their expression patterns, subcellular localization, E3 ligase activity, substrate specificity, and phenotypic effects when mutated or overexpressed.

What are common challenges when working with recombinant RING-H2 proteins and how can they be addressed?

Working with recombinant RING-H2 proteins like ATL32 can present several challenges:

• Protein solubility issues: RING-H2 proteins with transmembrane domains can be difficult to solubilize.

  • Solution: Use detergents like CHAPS or DDM; express truncated versions without the transmembrane domain; use fusion tags that enhance solubility (MBP, SUMO)

• Maintaining proper zinc coordination: The RING-H2 domain requires proper coordination of zinc ions for structural integrity.

  • Solution: Include zinc in purification buffers (typically 10-50 μM ZnCl₂); avoid strong chelating agents; use reducing agents to prevent oxidation of cysteine residues

• Protein instability: RING-H2 proteins may be prone to aggregation or degradation.

  • Solution: Optimize storage conditions (as detailed in section 3.2); use protease inhibitors during purification; determine optimal buffer conditions through thermal shift assays

• Proving E3 ligase activity: Demonstrating the enzymatic function can be challenging.

  • Solution: Test multiple E2 enzymes as partners; optimize reaction conditions (buffer, pH, temperature); use sensitive detection methods for ubiquitination; include positive controls

• Identifying physiologically relevant substrates: Distinguishing true substrates from non-specific interactions.

  • Solution: Validate interactions through multiple independent methods; perform competition assays; demonstrate specificity through mutagenesis of the RING-H2 domain

Careful optimization of experimental conditions and use of multiple complementary approaches can help overcome these challenges.

How can researchers verify the structural integrity of purified recombinant ATL32?

Verifying the structural integrity of purified recombinant ATL32 is essential to ensure that experimental results reflect the protein's native function. Several methods can be employed:

• Circular dichroism (CD) spectroscopy: To assess secondary structure elements and proper folding

• Thermal stability assays: Such as differential scanning fluorimetry (DSF) to determine the melting temperature and stability in different buffer conditions

• Metal content analysis: Using atomic absorption spectroscopy or inductively coupled plasma mass spectrometry (ICP-MS) to confirm the correct zinc:protein stoichiometry expected for a properly folded RING-H2 domain

• Limited proteolysis: To identify stably folded domains and flexible regions

• Size exclusion chromatography: To assess whether the protein exists as a monomer, forms oligomers, or aggregates

• NMR spectroscopy: For detailed structural analysis, particularly of the RING-H2 domain

• Functional assays: Such as E2 binding or auto-ubiquitination assays to confirm that the protein retains its expected biochemical activities

The combination of biophysical characterization and functional validation provides the most comprehensive assessment of structural integrity.

What evidence suggests roles for ATL family members in plant stress responses?

Several lines of evidence from the search results suggest roles for ATL family members in plant stress responses:

• ATL2 was identified as an early response gene, with transcript levels increasing rapidly after exposure to elicitors

• The transcript levels of ATL2 and ATL6 increase after treatment with cycloheximide, a translational inhibitor often used to study stress responses

• The structural features of ATL proteins, including the RING-H2 domain and transmembrane domain, suggest they may be involved in protein degradation during stress responses

• The ATL gene family is plant-specific, suggesting specialized functions in plant biology, potentially including adaptation to environmental stresses

• Arabidopsis deploys various signaling modules that perceive distinct stress signals, such as DNA damage and heat stresses . While not specifically mentioned in the search results, ATL32 may participate in these stress response pathways given its similarity to other ATL family members.

Further research is needed to establish the specific role of ATL32 in plant stress responses, but its membership in the ATL family suggests potential involvement in early stress signaling or downstream responses.

What is the relationship between ATL family proteins and plant development?

While the search results do not provide direct evidence for ATL32's role in plant development, we can infer potential relationships based on information about related ATL family members:

• Early response genes: ATL family genes like ATL2 have been identified as early response genes, which "often play pivotal roles during cell growth and differentiation" . This suggests potential involvement in developmental processes.

• Ubiquitin-mediated regulation: As putative E3 ubiquitin ligases, ATL family proteins likely regulate protein turnover, which is a critical aspect of developmental transitions and cellular differentiation.

• Potential integration with developmental pathways: The search results mention that in Arabidopsis, stress response pathways involving NAC-type transcription factors like ANAC044 and ANAC085 regulate cell cycle arrest . Since development and stress responses are often interconnected, ATL proteins may function at the interface of these processes.

Researchers interested in investigating ATL32's developmental roles could analyze expression patterns throughout plant development, examine phenotypes of knockout/overexpression lines during various developmental stages, and identify developmentally regulated proteins that interact with ATL32.

What are promising areas for future research on ATL32 and related proteins?

Based on the current understanding of ATL32 and related proteins, several promising research directions emerge:

• Comprehensive functional characterization: Determining the precise biological function of ATL32 through genetic, biochemical, and cellular approaches

• Stress-specific roles: Investigating whether ATL32 responds to specific types of stresses and how it contributes to stress tolerance

• Target identification: Systematic identification of ATL32 substrates under different conditions to understand its regulatory network

• Structural studies: Resolving the three-dimensional structure of ATL32, particularly the RING-H2 domain and its interaction with E2 enzymes

• Evolution of the ATL family: Comparative genomic and functional analyses across plant species to understand how this family evolved and diversified

• Integration with signaling networks: Determining how ATL32 connects with known stress response pathways, such as the ANAC044/ANAC085-Rep-MYB module

• Potential biotechnological applications: Exploring whether modification of ATL32 expression or activity could enhance stress tolerance in crops

Addressing these research questions would significantly advance our understanding of ATL32's role in plant biology and potentially contribute to developing more resilient crop varieties.

What cutting-edge technologies could advance our understanding of ATL32 function?

Several cutting-edge technologies could be applied to study ATL32 function in greater depth:

• CRISPR/Cas9 genome editing: For precise modification of ATL32 and related genes, creating knockout, knockin, and base-edited variants to assess function

• Proximity labeling proteomics: Using BioID or TurboID fused to ATL32 to identify proteins in close proximity in vivo, revealing potential substrates and interactors

• Single-cell transcriptomics: To understand cell-type specific expression patterns of ATL32 and responses to various stresses

• Advanced structural biology techniques: Including cryo-EM and integrative structural biology approaches to determine ATL32 structure, particularly in complex with E2 enzymes and substrates

• Optogenetics: Developing light-controlled versions of ATL32 to precisely manipulate its activity in specific cells and study temporal aspects of its function

• Synthetic biology approaches: Creating minimal systems to reconstitute ATL32 function in heterologous systems for mechanistic studies

• Multi-omics integration: Combining transcriptomics, proteomics, metabolomics, and phenomics to build comprehensive models of ATL32 function in plant stress responses

These technologies, used individually or in combination, could provide unprecedented insights into ATL32's molecular mechanism and biological function.

What is the current state of knowledge regarding ATL32 and what critical gaps remain?

The current state of knowledge regarding ATL32 indicates that it is a member of the plant-specific ATL family of RING-H2 proteins in Arabidopsis thaliana, likely functioning as an E3 ubiquitin ligase involved in protein degradation pathways. While its basic structural features and recombinant expression have been characterized, significant knowledge gaps remain regarding its specific biological functions, regulation, substrates, and integration into plant stress response networks.

Critical knowledge gaps that future research should address include:

• The specific stresses that regulate ATL32 expression and activity

• The identity of proteins targeted by ATL32 for ubiquitination

• The phenotypic consequences of ATL32 mutation or overexpression

• The E2 enzymes that partner with ATL32

• The subcellular localization and tissue-specific expression patterns

• The potential redundancy or specialization compared to other ATL family members