Recombinant Arabidopsis thaliana UPF0057 membrane protein At4g30650 (At4g30650)

What is At4g30650 and what are its basic structural properties?

At4g30650 is a UPF0057 membrane protein from Arabidopsis thaliana that belongs to the low temperature and salt responsive protein family. It consists of 73 amino acids with the sequence: MASNMEVFCEILIAILLPPLGVCLKRGCCTVEFLICLVLTILGYIPGIIYALYVIVFQNREGSTELGAPLNSA . The protein contains hydrophobic regions consistent with its classification as a membrane protein. Its UniProt ID is Q9M095, and it can be recombinantly expressed with various tags, most commonly an N-terminal His-tag for purification purposes .

What are the known physiological functions of At4g30650?

At4g30650 is primarily characterized as a low temperature and salt responsive protein, suggesting its involvement in abiotic stress response pathways . Transcriptional profiling studies have shown that this protein's expression is modulated under stress conditions, particularly in response to low temperature and high salinity. Additionally, research has demonstrated that At4g30650 expression is downregulated (fold change of -3.3) during ethylene signaling, indicating it may play a role in hormone-mediated stress responses . While the precise molecular mechanism remains under investigation, its membrane localization suggests it may function in stress signaling or membrane protection under adverse environmental conditions.

How is At4g30650 regulated at the transcriptional level?

At4g30650 expression is regulated by multiple environmental cues and signaling pathways. Transcriptomic analyses have revealed significant downregulation of At4g30650 (approximately 3.3-fold decrease) during ethylene signaling, suggesting that ethylene may suppress its expression as part of stress adaptation responses . In contrast, low temperature and salt stress appear to induce its expression, consistent with its annotation as a low temperature and salt responsive protein . These differential expression patterns indicate that At4g30650 is subject to complex transcriptional regulation that integrates multiple environmental inputs and hormone signaling networks.

What expression systems are optimal for recombinant At4g30650 production?

Based on available research, E. coli has been successfully used as an expression system for recombinant At4g30650 production . When expressing this membrane protein, considerations should include:

• Bacterial strain selection: BL21(DE3) or Rosetta strains are often preferred for membrane protein expression

• Temperature optimization: Lower temperatures (16-20°C) may improve proper folding

• Induction conditions: IPTG concentration and induction time require optimization

• Solubilization: As a membrane protein, detergents may be necessary for extraction

The small size (73 amino acids) makes this protein relatively amenable to bacterial expression compared to larger membrane proteins, but care must be taken to preserve the native conformation and functionality.

What purification strategies yield the highest purity of functional At4g30650?

The recommended purification strategy for His-tagged recombinant At4g30650 involves:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices as the primary purification step

• Washing with buffers containing low concentrations of imidazole to reduce non-specific binding

• Elution with higher concentrations of imidazole

• Further purification using size exclusion chromatography if higher purity is required

The final purified protein should achieve greater than 90% purity as determined by SDS-PAGE . For functional studies, care should be taken to maintain the membrane protein in appropriate detergent or lipid environments throughout purification to preserve its native structure and activity.

What are the optimal storage conditions for maintaining At4g30650 stability?

To maintain stability and activity of purified recombinant At4g30650, the following storage conditions are recommended:

• Store the lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution, add glycerol to a final concentration of 5-50% (optimally 50%) and aliquot for long-term storage at -20°C/-80°C

• For working stocks, store aliquots at 4°C for up to one week

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

• When reconstituting, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

The protein is typically stable in Tris/PBS-based buffer with 6% trehalose at pH 8.0 , which helps maintain protein integrity during storage and reconstitution.

What techniques are most effective for studying At4g30650 membrane localization?

For investigating the membrane localization of At4g30650, researchers should consider:

• Cellular fractionation followed by Western blotting: This approach can separate microsomal (membrane) fractions from cytosolic components, similar to techniques used in ethylene signaling studies with other Arabidopsis proteins

• Fluorescent protein fusions: GFP or YFP fusions expressed in plant protoplasts or whole plants

• Immunolocalization with specific antibodies: Using confocal microscopy to visualize the native protein's localization

• Membrane topology analysis: Protease protection assays or site-directed fluorescence labeling to determine orientation within the membrane

When designing these experiments, consider that At4g30650's small size (73 amino acids) may require careful fusion protein design to avoid disrupting trafficking or membrane insertion.

How can researchers assess At4g30650's role in stress response pathways?

To investigate At4g30650's role in stress responses, the following experimental approaches are recommended:

• Gene expression analysis:

  • qRT-PCR to monitor At4g30650 expression under various stress conditions (cold, salt, drought)

  • RNA-seq for genome-wide expression profiling in wild-type vs. knockout/knockdown lines

• Functional characterization:

  • Generate and phenotype knockout/knockdown lines under stress conditions

  • Complementation studies with the recombinant protein

  • Overexpression studies to assess enhanced stress tolerance

• Protein interaction studies:

  • Yeast two-hybrid or split-ubiquitin assays for membrane protein interactions

  • Co-immunoprecipitation with potential signaling partners

  • Bimolecular fluorescence complementation (BiFC) in planta

• Physiological assays:

  • Measure ion leakage, ROS production, or membrane integrity under stress conditions

  • Assess changes in stress hormone levels (ABA, ethylene) in response to At4g30650 manipulation

These approaches can help elucidate how At4g30650 functions within broader stress signaling networks, particularly in relation to ethylene response pathways where it shows significant differential expression .

How does At4g30650 integrate with ethylene and other hormone signaling networks?

Ethylene signaling appears to significantly influence At4g30650 expression, as evidenced by its downregulation (-3.3 fold change) in response to ethylene pathway activation . This integration can be investigated through:

• Epistasis analysis using:

  • Double mutants combining At4g30650 knockouts with mutations in ethylene signaling components (e.g., etr1, ein2, arr2)

  • Phenotypic characterization under hormone treatments and stress conditions

• Hormone crosstalk studies:

  • Examining At4g30650 expression in response to multiple hormones (ethylene, ABA, cytokinins)

  • Investigating how At4g30650 influences the balance between growth and stress responses

• Phosphorylation analysis:

  • Determining if At4g30650 is post-translationally modified in response to hormone signaling

  • Identifying kinases or phosphatases that may regulate its activity

The involvement of At4g30650 in ethylene responses may be particularly relevant to understanding how plants coordinate growth inhibition and stress tolerance mechanisms, as ethylene often mediates these opposing processes during environmental challenges.

What structural features of At4g30650 are critical for its function in stress response?

Understanding structure-function relationships for At4g30650 requires sophisticated analysis of this small membrane protein:

• Structural prediction and modeling:

  • Secondary structure prediction suggests multiple transmembrane domains

  • Homology modeling based on related UPF0057 family proteins

• Site-directed mutagenesis approaches:

  • Targeting conserved residues, particularly those in predicted functional domains

  • Examining the role of cysteine residues in potential disulfide bond formation

  • Investigating the importance of the C-terminal domain for protein-protein interactions

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • NMR studies for more detailed structural information

  • Lipid interaction studies to understand membrane association

• Functional reconstitution:

  • Incorporation into liposomes or nanodiscs for in vitro functional assays

  • Electrophysiology studies if ion channel or transporter activity is suspected

These approaches can help determine whether At4g30650 functions primarily through structural roles in membrane integrity during stress or through specific protein-protein interactions in signaling cascades.

How should researchers interpret conflicting data regarding At4g30650 function?

When encountering conflicting data about At4g30650 function, consider:

• Context-dependent effects:

  • Different plant tissues or developmental stages may show different functions

  • Environmental conditions during experiments may influence results

  • The protein may have distinct functions depending on stress severity or duration

• Methodological considerations:

  • Expression level differences between studies (overexpression vs. native levels)

  • Tag interference with protein function in recombinant studies

  • Differences in experimental systems (in vitro vs. in vivo, heterologous vs. native)

• Systematic analysis approach:

  • Create a comparison table of experimental conditions across studies

  • Evaluate genetic background differences that might explain phenotypic variations

  • Consider redundancy with other family members that may mask phenotypes

When contradictory results emerge, it's valuable to design experiments that directly test alternative hypotheses under identical conditions to resolve discrepancies.

What controls are essential when working with recombinant At4g30650?

Critical controls for experiments with recombinant At4g30650 include:

• Protein quality controls:

  • SDS-PAGE to confirm purity (>90% recommended)

  • Western blot with anti-His antibodies to verify identity

  • Mass spectrometry to confirm sequence integrity

  • Activity assays specific to hypothesized function

• Experimental controls:

  • Empty vector controls in expression studies

  • Heat-denatured protein controls to distinguish specific from non-specific effects

  • Wild-type protein compared to site-directed mutants

  • Vehicle controls matching reconstitution buffer composition

• Biological context controls:

  • Comparison with native protein behavior where possible

  • Complementation of knockout phenotypes to confirm functionality

  • Dose-response relationships to establish physiological relevance

Including these controls ensures that observations are specifically attributable to At4g30650 function rather than experimental artifacts or contaminants.

How might At4g30650 be utilized in developing stress-tolerant crops?

The potential applications of At4g30650 in agricultural biotechnology include:

• Transgenic approaches:

  • Overexpression in crop species to potentially enhance stress tolerance

  • Promoter modifications to optimize expression timing under stress conditions

  • Targeted expression in specific tissues most vulnerable to stress damage

• Marker-assisted breeding:

  • Identifying natural variants of At4g30650 orthologs in crop species

  • Screening for beneficial alleles associated with enhanced stress resilience

  • Developing molecular markers for efficient selection

• Genome editing strategies:

  • CRISPR/Cas9 modification of promoter regions to alter expression dynamics

  • Targeted amino acid changes to enhance protein stability or function

  • Modifying regulatory elements to optimize stress-responsive expression

• Experimental design considerations:

  • Field trials under varied stress conditions to assess real-world performance

  • Combined stress treatments to evaluate performance under complex environmental challenges

  • Yield component analysis to identify specific improvements in stress resilience

Before agricultural application, thorough characterization of potential unintended consequences on growth, development, and yield under non-stress conditions is essential.

What are the most promising directions for future At4g30650 research?

Future research on At4g30650 should consider:

• Systems biology approaches:

  • Integrating transcriptomics, proteomics, and metabolomics data

  • Network analysis to position At4g30650 within stress signaling pathways

  • Comparative studies across species to identify conserved functions

• Structural biology advancements:

  • Cryo-EM studies of the protein in membrane environments

  • Interaction studies with lipids and other membrane components

  • Dynamic structural changes during stress responses

• Mechanistic investigations:

  • Identification of direct interaction partners

  • Elucidation of post-translational modifications under stress

  • Determination of biochemical activities (potential enzymatic functions)

• Translational research:

  • Testing functions across diverse plant species

  • Evaluating potential as a biomarker for stress resilience

  • Engineering enhanced variants with improved function

These research directions would significantly advance our understanding of how small membrane proteins like At4g30650 contribute to plant stress adaptation and potentially lead to applications in agriculture and biotechnology.