Recombinant Arabidopsis thaliana Uncharacterized membrane protein At1g16860 (At1g16860)

What is known about the basic characteristics of At1g16860 protein?

At1g16860 is a 474-amino acid membrane protein from Arabidopsis thaliana (Mouse-ear cress), with UniProt accession number Q9FZ45 and UniProt ID Y1686_ARATH . The protein is encoded by the gene At1g16860, also known by the ORF name F6I1.14 . While it has been categorized as a membrane protein, its specific function remains uncharacterized, hence its designation as "Uncharacterized membrane protein At1g16860" .

How is recombinant At1g16860 protein typically produced for research?

Recombinant At1g16860 protein is commonly produced through heterologous expression in E. coli expression systems . The full-length protein (amino acids 1-474) is typically fused to an N-terminal His tag to facilitate purification . This tagged fusion protein is then expressed in E. coli, purified, and provided as a lyophilized powder for research purposes .

What is the optimal storage condition for recombinant At1g16860 protein?

According to product specifications, the optimal storage condition for recombinant At1g16860 protein is at -20°C/-80°C upon receipt, with aliquoting recommended for multiple use scenarios . The protein is typically stored in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . It's important to avoid repeated freeze-thaw cycles as this can compromise protein integrity . For working aliquots, storage at 4°C for up to one week is recommended .

How should recombinant At1g16860 protein be reconstituted for experimental use?

For reconstitution, it is recommended to briefly centrifuge the vial containing lyophilized protein prior to opening to ensure the contents are at the bottom . The protein should be reconstituted in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . To enhance stability, adding glycerol to a final concentration of 5-50% is recommended, with 50% being the standard concentration for long-term storage at -20°C/-80°C .

What post-translational modifications have been identified in At1g16860 protein?

Extensive phosphorylation has been documented for At1g16860 protein, with multiple phosphorylation sites identified through various studies and databases including p3DB, PhosPhAt, and UniProt . The following table summarizes some of the well-documented phosphorylation sites:

These multiple phosphorylation sites suggest that At1g16860 function may be highly regulated through phosphorylation, which is a common regulatory mechanism for membrane proteins involved in signaling pathways .

How can researchers effectively design experiments to study At1g16860 phosphorylation in vivo?

Given the multiple phosphorylation sites documented in At1g16860 , researchers investigating its phosphorylation in vivo should consider a multi-faceted approach:

• Phospho-specific antibodies: Design antibodies targeting the most frequently detected phosphorylation sites (S45, S74, S76, S95, S169) for use in Western blotting or immunoprecipitation experiments.

• Mass spectrometry: Implement LC-MS/MS analysis of immunoprecipitated At1g16860 from plant tissues to identify and quantify phosphorylation sites under different conditions or treatments.

• Phosphorylation site mutations: Generate transgenic Arabidopsis lines expressing At1g16860 with phospho-null mutations (S/T to A) or phospho-mimetic mutations (S/T to D/E) at key phosphorylation sites.

• Phos-tag SDS-PAGE: Employ this modified gel electrophoresis technique to separate phosphorylated from non-phosphorylated forms of the protein based on mobility shifts.

• Time-course experiments: Analyze phosphorylation dynamics following various stimuli (hormones, stresses, pathogens) to determine if At1g16860 phosphorylation is stimulus-dependent.

This comprehensive approach would provide insights into the physiological relevance of At1g16860 phosphorylation and potential regulatory mechanisms.

What are the current challenges in expressing and purifying membrane proteins like At1g16860 for structural studies?

Membrane proteins like At1g16860 present several challenges for expression and purification, particularly for structural studies:

• Expression system limitations: While E. coli is commonly used for At1g16860 expression , membrane proteins often face issues with proper folding, insertion into membranes, and toxicity to the host. Alternative systems such as yeast, insect cells, or cell-free systems may be necessary for optimal expression.

• Detergent selection complexity: As a membrane protein, At1g16860 requires careful selection of detergents for extraction and solubilization. Different detergents can affect protein stability, activity, and crystallization properties.

• Post-translational modification fidelity: The extensive phosphorylation observed in At1g16860 may not be properly reproduced in heterologous systems, potentially affecting function and structure.

• Protein stability issues: Membrane proteins are often unstable once removed from their native lipid environment, requiring optimization of buffer conditions, additives, and temperature.

• Yield limitations: Typically, membrane protein yields are lower than soluble proteins, requiring scale-up of expression systems.

Researchers should consider these challenges when designing purification strategies for At1g16860, particularly if structural studies are the ultimate goal.

How might At1g16860 protein purity affect experimental outcomes and what methods can ensure optimal purity?

Protein purity is critical for experimental outcomes, especially with an uncharacterized protein like At1g16860:

• Impact of impurities:

  • Contaminants may possess enzymatic activities that could be mistakenly attributed to At1g16860

  • Co-purifying proteins might interfere with interaction studies

  • Heterogeneity can prevent successful crystallization for structural studies

  • Endotoxins from E. coli expression systems can affect cell-based assays

• Methods to ensure optimal purity:

  • Multi-step purification strategy, combining IMAC (via the His-tag) with size exclusion and ion exchange chromatography

  • Detergent screening to optimize membrane protein extraction

  • Western blot verification using anti-His antibodies to confirm identity

  • Mass spectrometry analysis to verify protein identity and detect contaminants

  • Analytical size exclusion chromatography to assess homogeneity

  • Dynamic light scattering to detect aggregation

Current commercial preparations of recombinant At1g16860 typically achieve >90% purity as determined by SDS-PAGE , which is sufficient for many applications but may require further purification for structural or detailed biochemical studies.

What bioinformatic approaches can help predict potential functions of the uncharacterized At1g16860 protein?

For an uncharacterized protein like At1g16860, bioinformatic approaches can provide valuable functional predictions:

• Sequence-based predictions:

  • BLAST and PSI-BLAST searches to identify distant homologs with known functions

  • Multiple sequence alignments to identify conserved residues that might be functionally important

  • Analysis of the extensive phosphorylation sites for conserved kinase recognition motifs

• Structural predictions:

  • Transmembrane domain prediction using tools like TMHMM, TOPCONS, or CCTOP

  • Secondary structure prediction using tools like PSIPRED

  • 3D structure prediction using AlphaFold2 or RoseTTAFold

  • Binding site prediction to identify potential ligand interaction sites

• Systems biology approaches:

  • Co-expression analysis with genes of known function

  • Phylogenetic profiling to identify proteins with similar evolutionary patterns

  • Protein-protein interaction network predictions

• Subcellular localization prediction:

  • Signal peptide analysis

  • Organelle targeting sequence identification

  • Membrane topology prediction

• Functional domain analysis:

  • InterProScan to identify conserved domains

  • Motif scanning to identify short functional motifs

These complementary approaches can generate testable hypotheses about At1g16860 function, guiding experimental design and prioritization.

How can researchers design definitive experiments to elucidate the biological function of At1g16860?

For comprehensive functional characterization of At1g16860, researchers should implement a multi-faceted experimental strategy:

• Reverse genetics approach:

  • Generate CRISPR-Cas9 knockout lines for complete loss-of-function

  • Create knockdown lines using RNAi or artificial microRNA technology

  • Develop conditional knockout systems for essential genes

  • Design overexpression lines to assess gain-of-function phenotypes

  • Implement complementation studies with wild-type and mutated versions

• Phenotypic characterization under diverse conditions:

  • Growth parameters (root length, leaf size, flowering time)

  • Response to abiotic stresses (drought, salt, temperature extremes)

  • Response to biotic stresses (bacterial, fungal, viral pathogens)

  • Cellular phenotypes (membrane organization, subcellular structures)

• Spatiotemporal expression analysis:

  • Tissue-specific expression using promoter-reporter fusions

  • Developmental regulation using time-course analyses

  • Stimulus-responsive expression patterns

• Protein localization and dynamics:

  • Subcellular localization using fluorescent protein fusions

  • Membrane microdomain analysis (lipid rafts, nanodomains)

  • Protein turnover and trafficking studies

• Interactome mapping:

  • Split-ubiquitin membrane yeast two-hybrid screening

  • Co-immunoprecipitation coupled with mass spectrometry

  • Proximity labeling (BioID or APEX2) to identify neighboring proteins

This comprehensive approach would provide complementary lines of evidence to establish At1g16860's biological function.

What methodological approaches can resolve contradictory data in At1g16860 phosphorylation studies?

When facing contradictory phosphorylation data for At1g16860, researchers should consider:

• Technical validation approaches:

  • Compare different phospho-enrichment methods (TiO₂, IMAC, phospho-antibodies)

  • Implement parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) for targeted quantification

  • Validate key sites using orthogonal methods (Phos-tag, phospho-specific antibodies)

  • Assess biological variability through increased replication

• Resolution of site localization ambiguities:

  • Employ electron-transfer dissociation (ETD) or electron-capture dissociation (ECD) for improved site localization

  • Synthesize phosphopeptide standards for ambiguous sites

  • Generate phospho-null mutants to confirm site identity

• Context-dependent phosphorylation assessment:

  • Carefully control experimental conditions (developmental stage, tissue type, time of day)

  • Consider stimulus-dependent phosphorylation (duration and intensity of treatment)

  • Evaluate phosphatase inhibitor effects during sample preparation

• Integrative analysis:

  • Combine data from multiple studies using meta-analysis approaches

  • Develop confidence scores based on reproducibility across studies

  • Compare phospho-proteomics data with kinase consensus motifs

• Functional validation of conflicting sites:

  • Generate phospho-mimetic and phospho-null mutations for conflicting sites

  • Assess functional consequences in vivo and in vitro

  • Develop targeted assays for specific phosphorylation events

This methodical approach can help resolve contradictions and establish a more accurate phosphorylation profile of At1g16860.

How might researchers investigate potential roles of At1g16860 in plant immunity pathways?

Based on emerging connections between membrane proteins and plant immunity, researchers could investigate At1g16860's potential role through:

• Pathogen response profiling:

  • Challenge At1g16860 mutants with diverse pathogens (bacterial, fungal, viral)

  • Measure standard immunity markers (ROS burst, callose deposition, PR gene expression)

  • Assess susceptibility phenotypes quantitatively (bacterial growth, lesion size)

• Comparative analysis with known immunity components:

  • Analyze At1g16860 expression patterns in relation to ATG6 and NPR1, which are known to interact and enhance Arabidopsis resistance to Pst DC3000/avrRps4

  • Test for genetic interactions through double mutant analysis

  • Investigate potential physical interactions with immunity regulators

• Hormone signaling assessment:

  • Measure salicylic acid, jasmonic acid, and ethylene levels in At1g16860 mutants

  • Analyze expression of hormone-responsive genes

  • Test exogenous hormone sensitivity

• PAMP-triggered immunity evaluation:

  • Monitor responses to PAMPs (flg22, elf18, chitin)

  • Assess PTI marker gene expression

  • Evaluate signaling events (MAPK activation, calcium influx)

• Effector-triggered immunity assessment:

  • Test response to specific pathogen effectors

  • Analyze hypersensitive response development

  • Evaluate R-protein mediated signaling

• Phosphorylation dynamics during infection:

  • Monitor changes in At1g16860 phosphorylation status during pathogen challenge

  • Identify infection-specific phosphorylation sites

  • Determine kinases activated during immunity responses

This comprehensive approach would determine whether At1g16860 functions in plant immunity pathways and how it might relate to the ATG6-NPR1 interaction reported in the literature .

How can researchers determine if At1g16860 phosphorylation affects its interaction with other proteins or membrane localization?

To investigate the functional consequences of At1g16860 phosphorylation on protein interactions and localization:

• Phosphorylation-dependent interaction studies:

  • Perform comparative interactome analysis of wild-type At1g16860 versus phospho-null mutants

  • Implement phosphorylation-state specific protein complementation assays

  • Develop quantitative FRET-based assays to measure interaction affinities under different phosphorylation conditions

• Membrane microdomain association analysis:

  • Isolate membrane microdomains (detergent-resistant membranes) and compare At1g16860 distribution between wild-type and phospho-mutants

  • Employ super-resolution microscopy to visualize phosphorylation-dependent membrane clustering

  • Use FRAP (Fluorescence Recovery After Photobleaching) to measure membrane mobility changes upon phosphorylation

• Phosphorylation-dependent trafficking studies:

  • Track protein movement using photoconvertible fluorescent protein fusions

  • Implement pulse-chase experiments with phospho-mutants

  • Quantify endocytosis and recycling rates under different phosphorylation states

• Structural consequences of phosphorylation:

  • Employ hydrogen-deuterium exchange mass spectrometry to detect conformational changes

  • Use circular dichroism to assess secondary structure alterations

  • Implement crosslinking mass spectrometry to detect proximity changes

• Temporal dynamics analysis:

  • Develop phospho-specific biosensors to monitor phosphorylation in real-time

  • Implement optogenetic tools to spatiotemporally control kinase activity

  • Correlate phosphorylation events with localization changes using live-cell imaging

These approaches would provide mechanistic insights into how phosphorylation regulates At1g16860 function through altered protein interactions and subcellular localization.

What high-throughput screening approaches could identify small molecules that interact with At1g16860?

For identifying small molecules that interact with At1g16860, researchers could implement:

• In vitro binding assays:

  • Develop a thermal shift assay (differential scanning fluorimetry) to screen for compounds that stabilize At1g16860

  • Implement surface plasmon resonance (SPR) for direct binding detection

  • Use microscale thermophoresis (MST) to measure binding affinities in solution

• Functional screening approaches:

  • Design activity-based assays if potential enzymatic functions can be predicted

  • Develop yeast-based screening systems with growth readouts

  • Implement mammalian cell-based reporter assays

• Computational screening methods:

  • Structure-based virtual screening if structural data becomes available

  • Ligand-based approaches using compounds that affect similar proteins

  • Machine learning approaches trained on membrane protein-ligand interactions

• Phenotypic screening in planta:

  • Screen chemical libraries using At1g16860 overexpression or knockout lines

  • Look for compounds that rescue or phenocopy mutant phenotypes

  • Implement high-content imaging to detect subcellular localization changes

• Target identification validation:

  • Develop photoaffinity probes from hit compounds

  • Use cellular thermal shift assays (CETSA) to confirm direct binding

  • Generate resistant mutants to identify binding sites

• Structure-activity relationship development:

  • Synthesize analogs of hit compounds

  • Correlate chemical features with binding affinity or functional outcomes

  • Use medicinal chemistry approaches to optimize potency and selectivity

This comprehensive screening cascade would enable the identification of chemical probes that could be valuable tools for studying At1g16860 function and potentially lead to the development of compounds with agricultural applications.