Recombinant Arabidopsis thaliana Putative RING-H2 finger protein ATL50 (ATL50)

What is the basic structure and classification of ATL50 protein?

ATL50 belongs to the Arabidopsis Tóxicos en Levadura (ATL) family of RING-H2 finger proteins. These proteins typically contain a highly conserved RING-H2 finger domain that is essential for E3 ubiquitin ligase activity. The RING-H2 domain coordinates two zinc atoms in a cross-brace structure, which is characteristic of proteins involved in the ubiquitination pathway. As with other ATL family members, ATL50 likely contains a transmembrane domain at the N-terminus, followed by a basic region and the RING-H2 finger domain . The protein is primarily involved in targeted protein degradation through the ubiquitin-proteasome system, which is crucial for various cellular processes including abiotic stress responses.

How does ATL50 function in the ubiquitination pathway?

Like other ATL family E3 ligases such as ATL5, ATL50 likely functions as a substrate-recognition component in the ubiquitin-proteasome system. The protein would operate by:

• Recognizing specific substrate proteins for degradation

• Facilitating the transfer of ubiquitin from an E2 conjugating enzyme to the target substrate

• Enabling polyubiquitination of the target protein, marking it for proteasomal degradation

This process is critical for protein turnover and cellular homeostasis. Studies with ATL5 have demonstrated that these E3 ligases mediate the polyubiquitination and degradation of specific target proteins in a proteasome-dependent manner .

What cellular localization patterns are typical for ATL50?

Based on studies of similar ATL family proteins, ATL50 is likely localized to cellular membranes, particularly the endoplasmic reticulum or plasma membrane, due to its predicted transmembrane domain. The RING-H2 domain would extend into the cytoplasm, allowing interaction with cytosolic components of the ubiquitination machinery and target proteins. Subcellular localization studies would typically involve fluorescent protein fusions and confocal microscopy to visualize the precise distribution pattern within plant cells .

What are the optimal expression systems for producing recombinant ATL50?

For recombinant expression of ATL50, researchers should consider:

• Bacterial expression systems: E. coli BL21(DE3) strains are commonly used for initial expression trials, though membrane-associated proteins like ATL50 may present solubility challenges.

• Yeast expression systems: S. cerevisiae or P. pastoris can provide a eukaryotic environment with appropriate post-translational modifications.

• Plant-based expression systems: Transient expression in Nicotiana benthamiana or stable transformation of Arabidopsis cell cultures may yield more naturally folded protein.

For functional studies, expression constructs should preserve the critical RING-H2 domain integrity. When expressing the full protein proves challenging, researchers often focus on the catalytic RING-H2 domain alone. Expression should be validated using Western blot analysis with appropriate antibodies or epitope tags .

How can the E3 ligase activity of ATL50 be measured in vitro?

The E3 ligase activity of ATL50 can be assessed through in vitro ubiquitination assays that include:

• Components: Purified recombinant ATL50, E1 activating enzyme, appropriate E2 conjugating enzyme, ubiquitin (native or tagged), ATP, and potential substrate proteins.

• Detection methods: Western blot analysis using anti-ubiquitin antibodies to visualize ubiquitination of substrates, or fluorescently labeled ubiquitin for real-time monitoring.

• Controls: Include reactions lacking ATP or specific components to confirm specificity, and use mutated versions of the RING-H2 domain as negative controls.

The appearance of high-molecular-weight ubiquitinated products on Western blots indicates successful E3 ligase activity. Experiments should systematically test various E2 enzymes to identify the optimal partners for ATL50, as E3 ligases often show preference for specific E2 enzymes .

What approaches are effective for identifying ATL50 protein substrates?

Identifying the specific substrates of ATL50 requires multifaceted approaches:

• Yeast two-hybrid screening: This can identify potential protein-protein interactions, though may yield false positives and requires validation through secondary methods.

• Co-immunoprecipitation: Pulling down ATL50 protein complexes from plant tissue can identify interacting proteins, including potential substrates.

• Proteomics approaches: Quantitative proteomics comparing wild-type and atl50 mutant plants can identify proteins with altered abundance, potentially representing substrates.

• Bimolecular fluorescence complementation (BiFC): This can confirm protein interactions in planta and provide information about subcellular localization of these interactions.

Validation of potential substrates should include in vitro ubiquitination assays and assessment of substrate stability in wild-type versus atl50 mutant backgrounds. Similar approaches have been successful in identifying ABT1 as a substrate for ATL5 in Arabidopsis .

How does ATL50 expression respond to different abiotic stresses?

Based on studies of related ATL family members, ATL50 expression is likely regulated by various environmental stresses. Investigation would typically involve:

• Quantitative RT-PCR analysis: Measuring ATL50 transcript levels in plants exposed to different stresses (cold, drought, salt, heat, wounding, osmotic stress).

• Promoter-reporter fusion studies: Using the ATL50 promoter fused to GUS or luciferase to visualize expression patterns under different stress conditions.

• Temporal expression profiles: Assessing expression changes over time after stress imposition to identify rapid or delayed responses.

Similar RING-H2 finger proteins like ShATL78L show differential expression under various stresses, with particular upregulation under cold stress in stress-tolerant plant varieties . ATL50 may exhibit comparable stress-responsive expression patterns that provide clues to its functional roles.

What phenotypes are associated with ATL50 overexpression or knockout in stress conditions?

Phenotypic characterization of transgenic plants with altered ATL50 expression should focus on:

• Stress tolerance assessment: Analyzing survival rates, growth parameters, and physiological markers (e.g., electrolyte leakage, chlorophyll content, ROS accumulation) under various stresses.

• Detailed phenotypic analysis: Examining developmental stages particularly sensitive to stress, including seed germination, seedling establishment, vegetative growth, and reproductive development.

• Comparative phenotyping: Comparing atl50 knockout/knockdown lines with overexpression lines and wild-type controls to establish gene function.

Studies of related ATL proteins show that overexpression can enhance tolerance to abiotic stresses like cold, drought, and oxidative stress, while knockdown lines typically show increased sensitivity . If ATL50 functions similarly, researchers would expect enhanced stress tolerance in overexpression lines and compromised tolerance in knockout mutants.

How does ATL50 coordinate with plant hormone signaling during stress responses?

Exploring hormone interactions with ATL50 would involve:

• Expression analysis: Monitoring ATL50 transcript levels after treatment with different plant hormones (ABA, JA, SA, ethylene, auxin).

• Double mutant analysis: Creating and phenotyping plants with mutations in both ATL50 and key components of hormone signaling pathways.

• Hormone sensitivity tests: Assessing whether atl50 mutants show altered sensitivity to exogenous hormones.

Many RING-H2 finger proteins, including ATL family members, show hormone-responsive expression patterns and function at the intersection of hormone signaling and stress responses . Determining which hormone pathways interact with ATL50 would provide insights into its regulatory networks.

What transcription factors regulate ATL50 expression?

Understanding the transcriptional regulation of ATL50 would require:

• Promoter analysis: Computational identification of potential transcription factor binding sites in the ATL50 promoter.

• Yeast one-hybrid assays: Screening transcription factors that bind to the ATL50 promoter.

• Chromatin immunoprecipitation (ChIP): Confirming in vivo binding of candidate transcription factors to the ATL50 promoter.

Based on studies of related ATL proteins, RAV family transcription factors may be candidates for regulating ATL50. For example, RAV2 has been shown to bind to the promoter of ShATL78L and regulate its transcription in response to environmental conditions .

How do post-translational modifications affect ATL50 activity?

Post-translational modifications of ATL50 might include:

• Phosphorylation: Could modulate E3 ligase activity or substrate specificity.

• Self-ubiquitination: Many RING-type E3 ligases undergo auto-ubiquitination, regulating their own stability.

• Redox-based modifications: Zinc-coordinating cysteines in the RING domain might be susceptible to oxidative stress-induced modifications.

Investigation methods would include mass spectrometry to identify modifications, site-directed mutagenesis to assess their functional significance, and in vitro assays comparing native and modified versions of the protein. These modifications could serve as molecular switches controlling ATL50 activity in response to environmental signals.

What protein complexes does ATL50 participate in?

Elucidating ATL50 protein complexes would require:

• Affinity purification coupled with mass spectrometry: Identifying proteins that co-purify with tagged ATL50 from plant tissues.

• Size exclusion chromatography: Determining the native molecular weight of ATL50-containing complexes.

• Blue native PAGE: Preserving and visualizing protein complexes.

Studies with related ATL proteins suggest potential interactions with components of the ubiquitination machinery (E2 enzymes), substrate proteins, and regulatory proteins. For example, ShATL78L has been shown to interact with CSN5B, a component of the COP9 signalosome, which may regulate its activity in stress responses .

How does ATL50 function differ from other members of the ATL family?

Despite structural similarities, ATL family members likely have distinct functions through:

• Substrate specificity: Each ATL protein may target different substrate proteins for degradation.

• Expression patterns: Temporal and spatial expression differences may indicate specialized functions.

• Interaction partners: Different regulatory proteins and E2 enzymes may interact with specific ATL family members.

Comparative studies should include phylogenetic analysis, expression profiling, and substrate identification for multiple ATL proteins. ATL5, for example, specifically targets ABT1 for degradation to regulate seed longevity , while other ATL proteins may target different substrates for different biological processes.

What mechanisms determine the substrate specificity of ATL50?

Substrate recognition by ATL50 likely involves:

• Structural determinants: Specific domains or motifs outside the RING-H2 domain may mediate substrate binding.

• Recognition sequences: Specific amino acid sequences in substrate proteins may serve as degrons.

• Co-factors or adaptors: Additional proteins may facilitate substrate recognition.

Domain swapping experiments between different ATL family members could identify regions responsible for substrate specificity. Structural studies, including protein crystallography or cryo-EM, would provide insights into the molecular basis of substrate recognition .

How do developmental stage and tissue type influence ATL50 function?

The role of ATL50 across development and tissues requires:

• Tissue-specific expression analysis: Using RT-qPCR or promoter-reporter fusions to map expression across tissues and developmental stages.

• Tissue-specific phenotyping: Examining atl50 mutant phenotypes in different plant tissues and at different developmental stages.

• Tissue-specific complementation: Using tissue-specific promoters to express ATL50 in particular tissues of atl50 mutants.

Like other ATL family members, ATL50 may show constitutive expression across various tissues (roots, leaves, stems, flowers, fruits) but with tissue-specific functions mediated by the presence of different substrate proteins or regulatory factors .

How has the function of ATL50 evolved across different plant species?

Evolutionary analysis of ATL50 would involve:

• Comparative genomics: Identifying orthologs across plant species and analyzing sequence conservation.

• Functional complementation: Testing whether ATL50 orthologs from other species can complement atl50 mutants in Arabidopsis.

• Expression pattern comparison: Determining whether orthologs show similar stress-responsive expression patterns.

The ATL gene family has expanded in plants, likely through gene duplication events, leading to functional diversification. Studying the evolution of substrate specificity and stress responsiveness across species would provide insights into how this protein family has adapted to different environmental conditions .