Recombinant Arabidopsis thaliana RING-H2 finger protein ATL75 (ATL75)

How is recombinant ATL75 typically produced for research purposes?

Recombinant ATL75 is commonly expressed in E. coli expression systems. The full-length protein (amino acids 1-226) can be produced with an N-terminal His tag to facilitate purification. After expression, the protein is typically purified and prepared as a lyophilized powder. For optimal storage and handling:

• Upon receipt, briefly centrifuge the vial before opening

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

• Add glycerol (recommended final concentration of 50%) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• Store at -20°C/-80°C for longer-term storage or at 4°C for up to one week

What expression patterns does ATL75 show under different stress conditions?

While specific data for ATL75 is not directly provided in the search results, research on the similar RING-H2 finger protein ShATL78L from Solanum habrochaites offers valuable insights into potential expression patterns. Related ATL proteins show differential expression under various stress conditions:

For ATL75-specific research, these experimental approaches could be adapted to characterize its expression profile under various stress conditions.

How can protein-protein interactions of ATL75 be identified and validated?

To identify and validate protein-protein interactions of ATL75, researchers can employ a multi-technique approach:

• Yeast Two-Hybrid Screening:

  • Use the full-length ATL75 or specific domains as bait against an Arabidopsis cDNA library

  • Verify positive interactions through growth on selective media and β-galactosidase assays

  • Note: The transmembrane domain might interfere with nuclear localization; consider using truncated versions

• Co-Immunoprecipitation (Co-IP):

  • Express tagged versions of ATL75 (His-tag already available in recombinant systems)

  • Perform pull-down assays followed by mass spectrometry to identify interacting partners

  • Validate with reciprocal Co-IP experiments

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse ATL75 and candidate interactors to complementary fragments of fluorescent proteins

  • Transiently express in plant cells (protoplasts or Nicotiana benthamiana)

  • Analyze reconstituted fluorescence as indication of protein interaction

• Surface Plasmon Resonance (SPR) or Microscale Thermophoresis (MST):

  • Quantitatively measure binding affinities between purified ATL75 and candidate partners

  • Determine kinetic parameters of protein interactions

Based on studies of related proteins, CSN5B might be a candidate interactor worth investigating, as it interacts with ShATL78L in the regulation of abiotic stress responses .

What approaches can be used to investigate the role of ATL75 in plant stress tolerance?

To investigate ATL75's role in stress tolerance, researchers can implement the following experimental approaches:

• Genetic Modification Studies:

  • Generate ATL75 overexpression lines in Arabidopsis

  • Create knockout/knockdown lines using CRISPR-Cas9 or RNAi

  • Perform phenotypic analysis under various stress conditions (cold, drought, salt, oxidative stress)

  • Compare stress tolerance parameters (survival rate, growth, photosynthetic efficiency)

• Transcriptomic Analysis:

  • Conduct RNA-seq on wild-type versus ATL75 modified lines under control and stress conditions

  • Identify differentially expressed genes to reveal regulatory networks

  • Validate key findings with RT-qPCR

• Biochemical Characterization:

  • Assess ubiquitination activity of ATL75 in vitro

  • Identify target proteins for ubiquitination

  • Measure degradation rates of target proteins in vivo

• Promoter Analysis:

  • Clone the ATL75 promoter region and conduct promoter-reporter assays

  • Identify transcription factors that bind to the ATL75 promoter using yeast one-hybrid assays

  • Based on studies of related proteins, RAV2 might be a candidate transcription factor, as it binds to the promoter of ShATL78L

How does mutation rate and pattern in the ATL75 gene compare to general trends in the Arabidopsis genome?

Recent research on mutation patterns in Arabidopsis thaliana has revealed interesting trends that may apply to the ATL75 gene:

• Reduced Mutation Rate in Genic Regions:

  • Mutation frequency is reduced by approximately half inside gene bodies compared to non-genic regions

  • For essential genes, mutation frequency can be reduced by up to two-thirds

  • This suggests that ATL75, as a gene encoding a functional protein, likely experiences fewer mutations than surrounding non-genic DNA

• Epigenomic Influence on Mutation Patterns:

  • Over 90% of variance in genome-wide mutation patterns surrounding genes can be explained by epigenomic and physical features

  • These patterns accurately predict genetic polymorphisms in natural Arabidopsis accessions (r = 0.96)

  • For ATL75, its specific chromatin context and epigenetic marks would likely influence its mutation rate

• Selection Pressure and Mutation Rate:

  • Genes under stronger purifying selection tend to have lower mutation rates

  • The degree of functional constraint on ATL75 would influence its mutation frequency

  • Analysis of non-synonymous to synonymous mutation ratios can indicate selection pressure on ATL75

To specifically study mutation patterns in ATL75, researchers could:

• Analyze ATL75 sequences across multiple Arabidopsis accessions

• Compare mutation frequencies in ATL75 versus flanking regions

• Assess epigenetic marks in the ATL75 locus and correlate with mutation patterns

• Calculate Tajima's D for ATL75 to determine if observed polymorphism patterns align with mutation bias or selection

What computational approaches can predict ATL75 functional partners and regulatory networks?

Researchers can employ several computational methods to predict ATL75's functional partners and regulatory networks:

• Protein Structure Prediction and Docking:

  • Generate 3D models of ATL75 using AlphaFold or similar tools

  • Perform in silico docking with potential interaction partners

  • Analyze binding interfaces and energetics

• Co-expression Network Analysis:

  • Utilize publicly available transcriptome datasets to identify genes co-expressed with ATL75

  • Construct gene co-expression networks to predict functional associations

  • Look for enriched biological processes within the network

• Ortholog Analysis:

  • Compare ATL75 with characterized RING-H2 proteins like ShATL78L

  • Transfer functional annotations from well-studied orthologs

  • Identify conserved interaction partners across species

• Promoter Analysis:

  • Scan the ATL75 promoter for transcription factor binding sites

  • Compare with promoters of genes showing similar expression patterns

  • Predict upstream regulatory factors controlling ATL75 expression

• Phylogenetic Analysis:

  • Construct phylogenetic trees of ATL family proteins

  • Identify closely related proteins with known functions

  • Infer potential functional roles based on evolutionary relationships

What are the optimal conditions for expressing and purifying recombinant ATL75?

For optimal expression and purification of recombinant ATL75:

• Expression System Optimization:

  • E. coli BL21(DE3) is commonly used for expression of plant proteins

  • Consider using strains enhanced for disulfide bond formation (Origami) for proper folding of the RING domain

  • Test induction conditions: IPTG concentration (0.1-1.0 mM), temperature (16°C, 25°C, 37°C), and duration (4h vs. overnight)

• Protein Solubility Enhancement:

  • Express as fusion with solubility tags (MBP, SUMO) in addition to His-tag

  • Add low concentrations of zinc (10-50 μM ZnCl₂) to the growth medium to support RING domain folding

  • Consider co-expression with chaperones to improve folding

• Purification Protocol:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Include zinc (10 μM) in all buffers to maintain RING domain structure

  • Consider a second purification step (ion exchange or size exclusion chromatography)

  • Optimize buffer conditions to maintain protein stability (typically Tris/PBS-based buffer, pH 8.0)

• Storage Recommendations:

  • Add 5-50% glycerol (50% recommended) for long-term storage

  • Store as aliquots at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

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

How can the E3 ubiquitin ligase activity of ATL75 be assayed in vitro?

To assay the E3 ubiquitin ligase activity of ATL75 in vitro:

• Components Required:

  • Purified recombinant ATL75 protein

  • Commercial ubiquitination kit containing:

    • Ubiquitin (preferably fluorescently labeled for easier detection)

    • E1 ubiquitin-activating enzyme

    • E2 ubiquitin-conjugating enzymes (test a panel of different E2s)

    • ATP regeneration system

  • Potential substrate proteins (if known)

• Assay Protocol:

  • Combine all components in reaction buffer

  • Incubate at 30°C for 1-3 hours

  • Terminate reaction with SDS-PAGE sample buffer

  • Analyze by SDS-PAGE and Western blotting

• Detection Methods:

  • Western blotting with anti-ubiquitin antibodies

  • Fluorescence detection if using labeled ubiquitin

  • Mass spectrometry to identify ubiquitination sites

• Controls to Include:

  • Negative control: omit ATP (ubiquitination is ATP-dependent)

  • Negative control: use catalytically inactive ATL75 mutant (mutations in RING domain)

  • Positive control: known E3 ligase with confirmed activity

• Substrate Identification:

  • If substrates are unknown, perform in vitro ubiquitination assays using plant protein extracts

  • Identify ubiquitinated proteins by mass spectrometry

  • Confirm specific substrates with purified recombinant proteins

What strategies can be used to resolve contradictory findings about ATL75 function?

When faced with contradictory findings about ATL75 function, researchers can employ several strategies:

• Reconcile Methodological Differences:

  • Compare experimental conditions across studies (plant growth conditions, stress treatments, etc.)

  • Evaluate genetic backgrounds used (different Arabidopsis ecotypes may show varied responses)

  • Assess protein expression levels in different studies (overexpression vs. endogenous levels)

• Perform Comprehensive Phenotypic Analysis:

  • Generate multiple independent transgenic lines (overexpression, knockdown, knockout)

  • Test responses across various developmental stages

  • Evaluate multiple stress conditions and combinations

  • Quantify phenotypes using standardized methods

• Consider Genetic Redundancy:

  • Identify closely related ATL proteins that may have overlapping functions

  • Generate double or triple mutants to overcome redundancy

  • Perform complementation tests to confirm gene function

• Analyze Tissue-Specific and Subcellular Functions:

  • Use tissue-specific promoters to express ATL75 in different tissues

  • Determine subcellular localization and how it relates to function

  • Investigate if contradictory findings might reflect different functions in different tissues

• Apply Multiple Experimental Approaches:

  • Combine genetic, biochemical, and physiological approaches

  • Use both in vivo and in vitro systems

  • Employ emerging technologies like CRISPR-based techniques for precise gene editing

How can ATL75 be used as a tool to study the ubiquitin-proteasome system in plants?

ATL75, as a putative E3 ubiquitin ligase, can serve as a valuable tool for studying the ubiquitin-proteasome system in plants:

• Substrate Targeting System:

  • Engineer chimeric proteins fusing ATL75's RING domain with different substrate recognition domains

  • Create synthetic degradation systems for proteins of interest

  • Study specificity determinants in E3-substrate recognition

• Ubiquitination Dynamics Analysis:

  • Use fluorescently tagged ATL75 and potential substrates to visualize ubiquitination in real-time

  • Monitor protein degradation kinetics in various cellular compartments

  • Investigate how stress conditions alter ubiquitination dynamics

• Proteasome Inhibition Studies:

  • Compare phenotypes of ATL75 overexpression with and without proteasome inhibitors

  • Identify which effects of ATL75 are dependent on proteasomal degradation

  • Study protein turnover rates of ATL75 substrates

• E2-E3 Interaction Studies:

  • Map which E2 conjugating enzymes preferentially work with ATL75

  • Identify structural elements determining E2-E3 specificity

  • Engineer altered specificity variants for biotechnological applications

• Biomarker Development:

  • Develop assays using ATL75 expression as a biomarker for specific stress responses

  • Create reporter systems based on ATL75 promoter activity

  • Design biosensors using ATL75's substrate targeting mechanisms

What epigenetic factors influence ATL75 expression and how can they be studied?

Based on findings about mutation patterns and gene expression in Arabidopsis, several epigenetic factors likely influence ATL75 expression:

• DNA Methylation Analysis:

  • Perform bisulfite sequencing of the ATL75 promoter and gene body

  • Compare methylation patterns under different environmental conditions

  • Use demethylating agents (5-azacytidine) to test if ATL75 expression is affected

• Histone Modification Profiling:

  • Conduct ChIP-seq for histone marks associated with active (H3K4me3, H3K36me3) and repressive (H3K27me3, H3K9me2) chromatin

  • Analyze how these marks change under stress conditions

  • Correlate histone modifications with ATL75 expression levels

• Chromatin Accessibility:

  • Perform ATAC-seq or DNase-seq to determine chromatin accessibility at the ATL75 locus

  • Identify potential regulatory regions based on accessibility patterns

  • Study how accessibility changes during development or stress responses

• Non-coding RNA Involvement:

  • Screen for natural antisense transcripts or long non-coding RNAs associated with the ATL75 locus

  • Investigate potential miRNAs targeting ATL75 mRNA

  • Study how these regulatory RNAs respond to environmental signals

• Experimental Approaches:

  • Use mutants defective in epigenetic pathways to assess ATL75 expression

  • Apply pharmacological inhibitors of epigenetic enzymes

  • Create reporter constructs with various regions of the ATL75 promoter to identify epigenetically regulated elements

Studies of Arabidopsis genes indicate that epigenomic features explain over 90% of variance in genome-wide mutation patterns surrounding genes, suggesting that similar mechanisms may influence ATL75 expression and evolution .

What are common challenges in working with ATL75 and how can they be addressed?

Researchers working with ATL75 may encounter several challenges:

• Protein Solubility Issues:

  • Challenge: RING-H2 proteins may form inclusion bodies when overexpressed

  • Solution: Express at lower temperatures (16-20°C), use solubility tags, test different E. coli strains, or include zinc in growth media

• Functional Redundancy:

  • Challenge: Phenotypes may be masked by redundant ATL family proteins

  • Solution: Create multiple knockout lines, use tissue-specific promoters, or employ inducible systems to bypass developmental effects

• Transmembrane Domain Complications:

  • Challenge: The predicted transmembrane domain may cause localization or purification problems

  • Solution: Create truncated versions for specific applications, use appropriate detergents during purification, or employ membrane protein expression systems

• Substrate Identification Difficulties:

  • Challenge: Identifying true in vivo substrates can be challenging

  • Solution: Combine BioID or proximity labeling with immunoprecipitation and mass spectrometry; use stabilized mutants that trap ubiquitination intermediates

• Inconsistent Stress Responses:

  • Challenge: Stress response phenotypes may vary between experiments

  • Solution: Carefully control growth conditions, standardize stress application protocols, use appropriate controls, and perform experiments at multiple developmental stages

How can mutation analysis provide insights into ATL75 structure-function relationships?

Mutation analysis can reveal critical insights into ATL75 structure-function relationships:

• Domain-Specific Mutations:

  • RING Domain: Create point mutations in zinc-coordinating residues (C and H) to disrupt E3 ligase activity

  • Transmembrane Domain: Introduce mutations to alter membrane association or topology

  • N-terminal and C-terminal Regions: Generate truncations to identify regions important for substrate recognition or regulation

• Systematic Mutagenesis Approaches:

  • Alanine-scanning mutagenesis: Systematically replace amino acids with alanine to identify essential residues

  • Charge reversal mutations: Change charged residues to opposite charges to test electrostatic interactions

  • Conservation-guided mutations: Target residues conserved across ATL family members

• Experimental Validation Methods:

  • Express mutant variants in atl75 knockout backgrounds

  • Perform complementation assays to assess functionality

  • Use yeast two-hybrid or co-IP to test effects on protein-protein interactions

  • Conduct in vitro ubiquitination assays to measure activity of mutant proteins

• Advanced Structure-Function Analysis:

  • Correlate experimental findings with structural predictions from AlphaFold

  • Use molecular dynamics simulations to understand effects of mutations

  • Compare with characterized mutations in related ATL proteins

• Evolutionary Context:

  • Compare with natural variants in different Arabidopsis accessions

  • Identify positions under purifying or positive selection

  • Use this information to guide targeted mutagenesis