Recombinant Arabidopsis thaliana Peroxisomal membrane protein PEX14 (PEX14)

What is the function of PEX14 in Arabidopsis thaliana peroxisomes?

PEX14 is a critical component of the peroxisomal membrane in Arabidopsis thaliana that functions as an essential docking protein in the peroxisomal protein import machinery. Based on comparative studies with other organisms like Hansenula polymorpha, PEX14 serves as a crucial component for matrix protein import into peroxisomes . In Arabidopsis, PEX14 participates in the translocation of proteins containing peroxisomal targeting signals (PTS) across the peroxisomal membrane. This protein constitutes one of the core components of peroxisome biogenesis and interacts with other peroxins to facilitate proper peroxisome function and development in plant cells.

What peroxisomal processes are affected by PEX14 dysfunction in Arabidopsis?

PEX14 dysfunction in Arabidopsis impairs multiple peroxisomal processes due to defective protein import. Based on studies of peroxin mutants, PEX14 deficiency would likely affect:

• Fatty acid β-oxidation, resulting in defective seedling establishment

• Photorespiration, causing growth defects in light conditions

• Hormone metabolism, particularly jasmonic acid and auxin pathways

• Detoxification of reactive oxygen species

Similar to observations in Hansenula polymorpha pex14 mutants, where peroxisomal matrix proteins were mislocalized to the cytosol , Arabidopsis plants with PEX14 defects would likely show cytosolic accumulation of peroxisomal enzymes and corresponding metabolic deficiencies.

What expression systems are most effective for producing recombinant Arabidopsis thaliana PEX14?

For recombinant expression of Arabidopsis thaliana PEX14, bacterial expression systems can be employed with specific modifications to enhance solubility. Drawing from successful approaches used for other peroxins, such as the PEX4-PEX22 complex , the following expression strategy is recommended:

• Expression vector design: Clone the PEX14 coding sequence into a vector containing solubility-enhancing tags such as His6-MBP (hexahistidine-maltose binding protein) fusion at the N-terminus. This approach was successfully used for the PEX4-PEX22 complex .

• Host selection: Use E. coli strains optimized for membrane protein expression, such as C41(DE3) or Rosetta 2(DE3).

• Expression conditions: Induce with lower IPTG concentrations (0.1-0.5 mM) at reduced temperatures (16-18°C) to promote proper folding.

• Co-expression strategy: Consider co-expressing PEX14 with interacting partners to enhance stability, similar to the successful co-expression of PEX4 with the soluble domain of PEX22 .

Since membrane proteins like PEX14 can be challenging to express in soluble form, these modifications are crucial for successful recombinant production.

What purification strategy yields the highest purity of functional recombinant PEX14?

A multi-step purification approach is recommended for obtaining high-purity functional recombinant Arabidopsis thaliana PEX14:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using the His6 tag, with buffer conditions containing mild detergents (0.1% n-dodecyl-β-D-maltoside or 1% CHAPS) to maintain membrane protein solubility.

• Tag removal: Incorporate a specific protease recognition site (TEV or PreScission) between the fusion tag and PEX14, similar to the approach used for the PEX4-PEX22 fusion .

• Secondary purification: Size exclusion chromatography to separate monomeric from aggregated forms and remove remaining contaminants.

• Quality assessment: Analyze protein purity by SDS-PAGE and functional integrity through binding assays with known interaction partners.

This strategy, adapted from successful approaches with other peroxisomal membrane proteins , addresses the challenges specific to membrane protein purification while maintaining the functional integrity of PEX14.

How can researchers optimize the solubility of recombinant Arabidopsis PEX14 during expression and purification?

Optimizing solubility of recombinant Arabidopsis PEX14 requires specific strategies for membrane proteins:

• Truncation approach: Express only the soluble domains of PEX14, excluding the transmembrane regions, as was done successfully with PEX22 (using residues 111-283 without the transmembrane domain) .

• Detergent screening: Systematically test different detergents (DDM, CHAPS, Brij-35) at varying concentrations to identify optimal solubilization conditions.

• Fusion partners: Beyond MBP, test other solubility-enhancing fusion partners such as SUMO, GST, or NusA.

• Buffer optimization: Include stabilizing agents like glycerol (10-15%) and reducing agents (1-5 mM DTT or TCEP) to prevent aggregation.

• Co-expression with interacting partners: Express PEX14 with its natural binding partners to mimic physiological conditions and enhance stability.

These approaches can significantly improve the yield of properly folded, soluble recombinant PEX14, overcoming the challenges previously encountered with other peroxisomal membrane proteins .

What structural features of Arabidopsis PEX14 are critical for its interaction with other peroxins?

Based on structural studies of peroxins and comparative analysis, the critical structural features of Arabidopsis PEX14 likely include:

• N-terminal domain: Contains conserved hydrophobic regions that facilitate protein-protein interactions, similar to the well-conserved N-terminal regions observed in yeast Pex14p .

• Coiled-coil motifs: Mediate specific interactions with other peroxins and contribute to the formation of the protein import machinery complex.

• Membrane-anchoring regions: Enable proper localization to the peroxisomal membrane while positioning the functional domains for optimal interactions.

• Conserved binding pockets: Specific sites for recognition of peroxisomal targeting signals (PTS1 and PTS2) or their receptors.

Studies of the Hansenula polymorpha Pex14p revealed that the N-terminal half of the protein contains particularly well-conserved hydrophobic regions (amino acids 10-51 and 84-109) that do not form membrane-spanning α-helices but are crucial for protein interactions . Similar features in Arabidopsis PEX14 likely contribute to its functional interactions with the peroxisome biogenesis machinery.

What methods are most effective for studying PEX14 interactions with other peroxisomal proteins?

Multiple complementary approaches are recommended for comprehensive characterization of PEX14 interactions:

• Yeast two-hybrid (Y2H) assays: Effective for initial screening of potential interaction partners, similar to approaches used to study PEX4-PEX22 interactions .

• Co-immunoprecipitation (Co-IP): Using either native antibodies against PEX14 or epitope-tagged versions to pull down interaction complexes from plant extracts.

• Bimolecular Fluorescence Complementation (BiFC): For visualizing protein interactions in planta and determining their subcellular localization.

• Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC): For quantitative measurement of binding affinities between purified PEX14 and partner proteins.

• Chemical cross-linking coupled with mass spectrometry: To identify interaction interfaces and transient protein complexes.

• Proximity-dependent biotin identification (BioID): For identifying proteins in close proximity to PEX14 in the cellular context.

These methods, when used in combination, provide robust data on both the specificity and dynamics of PEX14 interactions with other components of the peroxisomal protein import machinery.

How does post-translational modification affect PEX14 function in Arabidopsis?

Post-translational modifications (PTMs) of Arabidopsis PEX14 likely play critical roles in regulating its function:

• Ubiquitination: Given that the peroxisomal membrane-associated ubiquitination machinery (including PEX4-PEX22) is well-characterized in Arabidopsis , PEX14 may be regulated through ubiquitination. This modification could influence PEX14 stability, localization, or interaction capabilities.

• Phosphorylation: Potential phosphorylation sites in PEX14 might regulate its interaction with other peroxins or modulate its activity in response to cellular signaling pathways.

• Redox modifications: Considering the oxidative environment of peroxisomes, cysteine residues in PEX14 might undergo oxidation/reduction, affecting protein structure and function.

Experimental approaches to study these modifications include:

• Immunoprecipitation followed by mass spectrometry to identify PTMs

• Site-directed mutagenesis of modified residues to assess functional consequences

• In vitro modification assays with purified enzymes (e.g., kinases, E3 ligases)

• Phospho-specific or ubiquitin-specific antibodies for detection of modified forms

Understanding these modifications is crucial for elucidating the dynamic regulation of peroxisomal protein import in plants.

How can CRISPR-Cas9 gene editing be optimized for studying PEX14 function in Arabidopsis?

Optimizing CRISPR-Cas9 gene editing for PEX14 functional studies in Arabidopsis requires careful consideration of several factors:

• gRNA design strategy:

  • Target conserved functional domains based on comparative analysis with yeast PEX14

  • Design multiple gRNAs to create a series of variants with different functional defects

  • Use tools like CRISPR-P or CHOPCHOP optimized for Arabidopsis genome

• Conditional knockout approaches:

  • Complete loss of PEX14 may be lethal, similar to several peroxin mutations in Arabidopsis

  • Implement inducible or tissue-specific CRISPR systems

  • Consider creating an allelic series of mutations with varying severity

• Editing verification protocol:

  • PCR-based genotyping followed by sequencing

  • Western blotting to confirm protein expression changes

  • Peroxisome visualization using fluorescent markers to assess organelle integrity

• Phenotypic analysis framework:

  • Assess seedling establishment on media without sucrose

  • Measure photosynthetic parameters affected by photorespiration

  • Evaluate responses to environmental stresses

  • Analyze peroxisomal matrix protein localization using fluorescent reporters

This systematic approach enables precise dissection of PEX14 function while accommodating potential developmental essentiality of this peroxin.

What are the most informative experimental approaches for studying the dynamics of PEX14-mediated protein import in vivo?

For investigating PEX14-mediated protein import dynamics in living plant cells, the following approaches are most informative:

• Live-cell imaging with photoconvertible/photoswitchable fluorescent proteins:

  • Fuse peroxisomal matrix proteins with Dendra2 or mEos

  • Photoconvert proteins in the cytosol and track their import into peroxisomes

  • Measure import rates under different conditions or genetic backgrounds

• FRAP (Fluorescence Recovery After Photobleaching) analysis:

  • Bleach fluorescently-tagged peroxisomal proteins and measure recovery kinetics

  • Compare import rates between wild-type and PEX14 variant plants

  • Assess the impact of environmental conditions on import efficiency

• Single-molecule tracking:

  • Utilize super-resolution microscopy (e.g., PALM or STORM)

  • Track individual cargo proteins during the import process

  • Identify rate-limiting steps and transient interactions

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence imaging with ultrastructural analysis

  • Visualize the precise localization of PEX14 at the peroxisomal membrane

  • Examine structural changes associated with protein import

• Optogenetic tools for temporal control:

  • Develop light-inducible PEX14 variants to control import activity

  • Create optogenetically regulated cargo proteins

  • Study the kinetics of import initiation and completion

These approaches provide unprecedented temporal and spatial resolution of the PEX14-dependent protein import process, revealing mechanisms that cannot be captured by traditional biochemical methods.

How can comparative proteomics be used to understand the consequences of PEX14 dysfunction in different plant tissues?

Comparative proteomics offers powerful insights into the tissue-specific consequences of PEX14 dysfunction:

• Experimental design strategy:

  • Compare wild-type, pex14 knockdown/knockout, and complemented lines

  • Analyze multiple tissues: leaves, roots, seedlings, and reproductive organs

  • Include subcellular fractionation to distinguish peroxisomal, cytosolic, and other compartments

• Sample preparation protocol:

  • Optimize peroxisome isolation methods for different tissues

  • Employ sequential extraction to capture membrane-associated proteins

  • Use LOPIT (localization of organelle proteins by isotope tagging) for protein localization mapping

• Quantitative proteomics approaches:

  • TMT or iTRAQ labeling for multiplexed quantitative analysis

  • SILAC labeling in cell culture studies

  • Label-free quantification for broad protein coverage

• Data analysis framework:

  • Identify mislocalized peroxisomal matrix proteins in cytosolic fractions

  • Detect compensatory changes in related metabolic pathways

  • Map tissue-specific differences in response to PEX14 dysfunction

  • Analyze post-translational modifications of affected proteins

• Validation methods:

  • Western blotting for key peroxisomal proteins

  • Activity assays for peroxisomal enzymes

  • Microscopy to confirm protein mislocalization

  • Metabolomic analysis to correlate protein changes with metabolic outcomes

This comprehensive approach reveals not only the primary effects of PEX14 dysfunction on peroxisomal protein import but also secondary adaptations and tissue-specific consequences.

What strategies can address poor expression of recombinant Arabidopsis PEX14 in heterologous systems?

Poor expression of recombinant Arabidopsis PEX14 can be addressed through several targeted strategies:

• Codon optimization approach:

  • Optimize the PEX14 coding sequence for the expression host

  • Focus particularly on rare codons at the 5' end of the transcript

  • Consider using specialized strains (e.g., Rosetta) that supply rare tRNAs

• Expression construct redesign:

  • Create a fusion protein with highly soluble partners (MBP, SUMO, Trx)

  • Express truncated versions lacking problematic regions

  • Try dual expression systems similar to the PEX4-PEX22 approach

• Induction protocol modifications:

  • Reduce culture temperature to 15-18°C during induction

  • Use lower inducer concentrations (0.1-0.2 mM IPTG)

  • Extend expression time (24-48 hours)

  • Add chemical chaperones like 4% ethanol or 1% sorbitol to culture medium

• Host strain considerations:

  • Test multiple E. coli strains designed for membrane proteins

  • Consider alternative expression systems (insect cells, yeast)

  • Use strains with enhanced membrane protein folding capabilities

• Media and growth optimizations:

  • Use auto-induction media for gentler protein expression

  • Add membrane protein expression enhancers (e.g., betaine, sorbitol)

  • Optimize culture aeration and mixing conditions

These modifications can significantly improve expression levels of challenging membrane proteins like PEX14, as demonstrated with other peroxins that were initially difficult to express heterologously .

How can researchers distinguish between direct and indirect effects when analyzing PEX14 mutant phenotypes?

Distinguishing direct from indirect effects in PEX14 mutant phenotypes requires a multi-faceted approach:

• Time-course analysis:

  • Study the temporal progression of phenotypes after inducible disruption of PEX14

  • Primary effects typically manifest earlier than secondary consequences

  • Use high-temporal-resolution transcriptomics and metabolomics

• Complementation strategies:

  • Perform domain-specific complementation with truncated or chimeric PEX14 variants

  • Utilize tissue-specific or inducible expression systems

  • Complement with orthologs from other species to identify conserved functions

• Genetic interaction mapping:

  • Create double mutants with other peroxin genes

  • Conduct suppressor screens to identify genes that ameliorate pex14 phenotypes

  • Use synthetic genetic array analysis to identify genetic interactions

• Direct biochemical validation:

  • Perform in vitro binding assays with purified components

  • Use reconstitution experiments in artificial membrane systems

  • Conduct pulse-chase experiments to track protein import kinetics

• Comparative analysis framework:

  • Compare phenotypes across multiple pex mutants affecting different aspects of peroxisome function

  • Identify phenotypes unique to pex14 versus those common to all import-defective mutants

  • Utilize mathematical modeling to predict direct versus cascade effects

This systematic approach allows researchers to build a causal network from PEX14 dysfunction to observed phenotypes, distinguishing primary molecular defects from downstream consequences.

What are the most effective controls for validating the specificity of PEX14 antibodies in immunolocalization studies?

Proper validation of PEX14 antibodies for immunolocalization requires comprehensive controls:

• Genetic controls:

  • Perform immunolabeling in pex14 knockout/knockdown tissues

  • Use tissues overexpressing PEX14 to confirm signal enhancement

  • Include tissues expressing epitope-tagged PEX14 for parallel detection

• Biochemical validation:

  • Conduct Western blots on subcellular fractions to confirm specificity

  • Perform antibody pre-absorption with purified antigen

  • Test multiple antibodies raised against different PEX14 epitopes

• Co-localization controls:

  • Co-label with established peroxisomal membrane markers

  • Perform double immunolabeling with antibodies against known PEX14 interactors

  • Use fluorescent protein-tagged PEX14 as reference for antibody labeling pattern

• Technical controls:

  • Include secondary antibody-only controls

  • Implement peptide competition assays to demonstrate specificity

  • Test cross-reactivity with closely related proteins

• Method-specific controls:

  • For immunogold EM: include quantification of gold particle density in peroxisomes versus other compartments

  • For immunofluorescence: implement standardized image acquisition and analysis parameters

  • For super-resolution microscopy: include drift correction and channel alignment controls

These controls ensure that observed signals genuinely represent PEX14 localization rather than experimental artifacts, which is particularly important for membrane proteins that can be challenging for immunological detection.

What statistical approaches are most appropriate for analyzing PEX14 colocalization with other peroxisomal proteins?

For rigorous analysis of PEX14 colocalization with other peroxisomal proteins, the following statistical approaches are recommended:

Table 1: Comparison of Colocalization Analysis Methods for PEX14 Studies

Recommended analysis workflow:

• Apply appropriate background subtraction and noise filtering

• Use multiple complementary coefficients (both Pearson's and Manders')

• Implement Costes' randomization to determine statistical significance

• Perform object-based analysis when studying heterogeneous peroxisome populations

• Analyze a statistically significant number of cells (>30) across multiple biological replicates

This comprehensive approach provides robust quantification of PEX14 colocalization patterns while accounting for the specific challenges of peroxisomal imaging.

How can researchers integrate transcriptomic, proteomic, and metabolomic data to understand the systems-level impact of PEX14 dysfunction?

Integrating multi-omics data for understanding PEX14 dysfunction requires a systematic approach:

• Data preprocessing framework:

  • Normalize data within each omics platform

  • Address missing values appropriately for each data type

  • Harmonize sample identifiers across platforms

  • Apply batch correction methods when necessary

• Multi-omics integration strategies:

  • Correlation networks: Identify coordinated changes across datasets

  • Pathway enrichment analysis: Map changes to known biological pathways

  • Knowledge-based integration: Use protein-protein interaction networks as scaffolds

  • Machine learning approaches: Identify patterns distinguishing normal from dysfunctional states

• Causal relationship identification:

  • Time-course experiments to establish temporal order of changes

  • Bayesian network analysis to infer directional relationships

  • Comparative analysis with other pex mutants to identify PEX14-specific effects

• Visualization approaches:

  • Multi-dimensional scaling or t-SNE plots for global data structure

  • Pathway visualization with multiple data types overlaid

  • Clustered heatmaps showing coordinated changes across platforms

• Validation experiments:

  • Target validation of key nodes identified through integration

  • Perturbation experiments to test predicted regulatory relationships

  • Flux analysis to confirm metabolic pathway alterations

This integrated analysis allows researchers to build comprehensive models connecting PEX14 dysfunction to molecular changes and ultimately to physiological phenotypes, revealing both direct consequences and adaptive responses.

What are the most promising approaches for studying PEX14 regulation during plant development and stress responses?

Several cutting-edge approaches show promise for elucidating PEX14 regulation during development and stress:

• Single-cell omics approaches:

  • Single-cell RNA-seq to map cell type-specific PEX14 expression patterns

  • Single-cell proteomics to detect protein-level changes

  • Spatial transcriptomics to visualize expression in tissue context

• Developmental time-course analysis:

  • Stage-specific sampling during key developmental transitions

  • Tissue-specific promoter:reporter constructs to visualize dynamic regulation

  • Inducible expression systems to manipulate PEX14 levels at defined stages

• Stress-responsive regulatory elements identification:

  • Chromatin immunoprecipitation to identify transcription factors regulating PEX14

  • Promoter deletion analysis to map functional regulatory elements

  • DNase-seq or ATAC-seq to assess chromatin accessibility changes

• Post-translational regulation mechanisms:

  • Targeted mass spectrometry to identify and quantify PTMs

  • Proximity labeling to map the changing PEX14 interactome during stress

  • In vivo protein lifetime measurements using tandem fluorescent timers

• Systems biology modeling:

  • Mathematical modeling of peroxisome proliferation during stress

  • Agent-based models of peroxisome dynamics in different cell types

  • Flux balance analysis to predict metabolic consequences of PEX14 regulation

These approaches, especially when applied in combination, will provide unprecedented insights into how plants modulate peroxisome function through PEX14 regulation during both normal development and stress adaptation.

How might synthetic biology approaches be applied to engineer modified PEX14 proteins with enhanced or novel functions?

Synthetic biology offers exciting possibilities for PEX14 engineering:

• Domain swapping and chimeric proteins:

  • Create chimeras between plant and yeast PEX14 to identify functional domains

  • Swap interaction domains to alter binding partner specificity

  • Develop split-PEX14 systems for conditional peroxisome biogenesis

• Stimulus-responsive PEX14 variants:

  • Light-switchable domains to control peroxisome import

  • Temperature-sensitive variants for temporal regulation

  • Chemical-inducible systems for experimental control

• Enhanced import machinery:

  • Optimize binding interfaces based on structural data

  • Increase processivity through rational design

  • Reduce non-productive interactions through surface engineering

• Novel targeting capabilities:

  • Expand cargo recognition to non-natural substrates

  • Engineer PEX14 to recognize alternative targeting signals

  • Create programmable import systems for biotechnology applications

• Synthetic peroxisome design:

  • Minimal peroxisome systems with engineered PEX14 variants

  • Repurposing peroxisomes for novel metabolic pathways

  • Development of synthetic organelles with PEX14-inspired import systems

These synthetic approaches not only provide tools for fundamental research but also open possibilities for engineering plants with enhanced metabolic capabilities, stress resistance, or novel biosynthetic pathways through peroxisome modification.

What computational approaches can predict the impact of PEX14 mutations on protein import efficiency?

Advanced computational approaches can predict mutation impacts on PEX14 function:

• Structural modeling and molecular dynamics:

  • Homology modeling based on available peroxin structures

  • Molecular dynamics simulations to predict conformational changes

  • Binding interface analysis to identify critical residues

  • Free energy calculations to quantify mutation effects on stability

• Machine learning prediction tools:

  • Trained on existing peroxin mutation data

  • Feature extraction from sequence and structural properties

  • Ensemble methods combining multiple predictors

  • Deep learning approaches utilizing protein language models

• Network analysis methods:

  • Protein-protein interaction network perturbation modeling

  • Identification of critical nodes in the peroxisome import network

  • Robustness analysis to predict system-level impacts

• Evolutionary approaches:

  • Conservation analysis across species

  • Coevolution detection between interacting residues

  • Evolutionary coupling analysis to identify functionally linked positions

• Systems modeling of import kinetics:

  • Ordinary differential equation models of the import process

  • Parameter sensitivity analysis to identify rate-limiting steps

  • Stochastic simulations to capture variability in import efficiency

These computational approaches, particularly when integrated with experimental validation, provide powerful tools for predicting mutation consequences and designing rational modifications to enhance or alter PEX14 function in plant systems.