Recombinant Arabidopsis thaliana Protein cornichon homolog 5 (At4g12090)

What is the Arabidopsis thaliana Protein Cornichon Homolog 5?

Arabidopsis thaliana Protein Cornichon Homolog 5 (At4g12090) is a member of the CORNICHON family proteins that function as endoplasmic reticulum (ER) cargo receptors. It is a 135-amino acid transmembrane protein encoded by the At4g12090 gene, also annotated as F16J13.160, with UniProt ID Q9SZ74. The protein is characterized by a cornichon motif and an "IFRTL"-like Sec24-interacting motif, which are essential for its function in protein trafficking from the ER to the plasma membrane .

How many CORNICHON homologs are present in Arabidopsis thaliana?

Arabidopsis thaliana contains five CORNICHON homologs, designated as AtCNIH1 through AtCNIH5. These proteins share structural features including the cornichon motif and Sec24-interacting motifs. RNA of all AtCNIHs has been detected in pollen, with AtCNIH4 showing the highest expression in this tissue . Each CNIH protein appears to have specialized functions in different tissues and developmental stages.

What is the recommended protocol for reconstituting recombinant At4g12090 protein?

For optimal reconstitution of lyophilized recombinant At4g12090 protein:

• Centrifuge the vial briefly to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is recommended for optimal stability)

• Aliquot for long-term storage at -20°C/-80°C to avoid repeated freeze-thaw cycles

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

How does AtCNIH5 function in phosphate homeostasis?

AtCNIH5 functions as a phosphate (Pi) deficiency-induced ER cargo receptor that regulates Pi homeostasis through multiple mechanisms:

• Cargo selection and trafficking: AtCNIH5 facilitates the efficient ER export of PHOSPHATE TRANSPORTER 1 proteins (PHT1s) to the plasma membrane

• Interaction with AtPHF1: AtCNIH5 interplays with PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (AtPHF1) to promote PM targeting of AtPHT1s in a cell type-dependent manner

• Cell-type specificity: The protein is preferentially expressed in the outer root cell layers above the meristem

• Localization: AtCNIH5 localizes adjacent to the AtSEC16A-labeled ER exit sites

• Impact on Pi levels: Loss of AtCNIH5 confers reduced shoot Pi levels and decreased AtPHT1s but increased AtPHF1

Interestingly, AtCNIH5 has been found to increase in the root of the Pi overaccumulator pho2, suggesting a complex regulatory relationship in Pi signaling networks .

What other cargo proteins does AtCNIH5 interact with beyond PHT1 transporters?

Proteomic analysis has revealed that AtCNIH5 interacts with multiple membrane proteins beyond PHT1 transporters:

Additionally, proteomic analysis identified downregulated enzymes in cnih5 mutants that catalyze the biosynthesis of very long-chain fatty acids and nucleotide sugars for cell walls, suggesting AtCNIH5 may also facilitate the trafficking of these biosynthetic enzymes .

How does the cargo selection mechanism of AtCNIH5 differ from other CORNICHON proteins?

AtCNIH5 employs a distinct cargo selection mechanism compared to other CORNICHON proteins:

• C-terminal acidic residue independence: Unlike fungal and rice CNIHs that interact with their cognate cargoes through the C-terminal acidic motif, AtCNIH5's C-terminal acidic residue is not required for interaction with AtPHT1;1

• Cargo specificity: AtCNIH5 appears to have evolved specialized cargo recognition for phosphate transporters and related proteins involved in Pi starvation response

• Tissue specificity: AtCNIH5 shows differential expression patterns compared to other AtCNIHs, with particular upregulation in response to Pi limitation in root tissues

This unique mechanism may represent an evolutionary adaptation specific to the regulation of Pi homeostasis in Arabidopsis.

What are the optimal conditions for expressing recombinant At4g12090 protein?

The optimal conditions for expressing recombinant At4g12090 protein in E. coli include:

• Expression system: E. coli BL21(DE3) or equivalent strain

• Vector construction: Insert At4g12090 cDNA (encoding amino acids 1-135) into a pET-series vector with an N-terminal His-tag

• Induction conditions:

  • Culture temperature: 30°C pre-induction, 25°C post-induction

  • IPTG concentration: 0.5-1.0 mM

  • Induction time: 4-6 hours

• Harvest and lysis:

  • Resuspend cells in Tris/PBS-based buffer (pH 8.0)

  • Add protease inhibitors

  • Lyse cells by sonication or mechanical disruption

• Purification:

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

  • Elute with imidazole gradient (50-250 mM)

  • Verify purity by SDS-PAGE (>90%)

What methods are effective for studying AtCNIH5 interactions with cargo proteins?

Several complementary methods have proven effective for studying AtCNIH5 interactions with cargo proteins:

• Yeast split-ubiquitin assay:

  • Enables detection of membrane protein interactions

  • Fusion of N-terminal and C-terminal ubiquitin fragments to potential interacting partners

  • Positive interaction reconstitutes functional ubiquitin, releasing a transcription factor

• In-planta tripartite split-GFP assay:

  • Uses three fragments of GFP (GFP1-9, GFP10, GFP11)

  • Allows visualization of protein interactions in native plant cells

  • Can detect interactions at specific subcellular locations

• Co-immunoprecipitation (Co-IP):

  • Uses antibodies specific to AtCNIH5 or epitope tags

  • Can detect endogenous protein interactions

  • Western blot analysis confirms specific interactions

• Bimolecular fluorescence complementation (BiFC):

  • Split YFP/GFP fragments fused to potential interacting partners

  • Enables visualization of interactions in living cells

  • Provides spatial information about interaction sites

How can I design a CRISPR-Cas9 system to generate AtCNIH5 mutants?

To design an effective CRISPR-Cas9 system for generating AtCNIH5 mutants:

• sgRNA design:

  • Target early exons to ensure protein disruption

  • Select target sites with minimal off-target effects

  • Recommended target: 5' region of the coding sequence with NGG PAM site

  • Use CRISPR design tools (e.g., CRISPR-P 2.0) to identify optimal sgRNA sequences

• Vector construction:

  • Clone sgRNA into a plant-compatible CRISPR-Cas9 vector (e.g., pHEE401E)

  • Include plant-optimized Cas9 and appropriate selection markers

  • Validate construct by sequencing

• Transformation:

  • Use Agrobacterium-mediated floral dip transformation for Arabidopsis

  • Screen T1 transformants on selection media

  • Confirm editing by PCR and sequencing of the target region

• Mutation verification:

  • Use T7 endonuclease I assay or direct sequencing to identify mutations

  • Confirm homozygous mutants in T2 or T3 generations

  • Verify loss of AtCNIH5 expression by RT-PCR and Western blotting

How do I interpret phenotypic differences between wild-type and cnih5 mutant plants?

When interpreting phenotypic differences between wild-type and cnih5 mutant plants, consider the following analytical approach:

• Growth parameters analysis:

  • Measure root length, lateral root number, shoot biomass, and leaf area

  • Compare growth under both Pi-replete and Pi-limited conditions

  • Analyze data using appropriate statistical tests (ANOVA with post-hoc comparisons)

  • Pay special attention to root architecture changes, as AtCNIH5 is highly expressed in root tissues

• Phosphate content measurement:

  • Quantify Pi content in shoots and roots separately

  • Normalize to fresh or dry weight

  • Compare Pi distribution between tissues

  • The cnih5 mutant typically shows reduced shoot Pi levels

• Expression analysis of Pi starvation response genes:

  • Perform qRT-PCR for PHT1 family genes and other Pi-responsive genes

  • Use housekeeping genes as internal controls

  • Calculate fold-change relative to wild-type

  • Correlate gene expression changes with phenotypic differences

• Protein localization assessment:

  • Use fluorescently tagged PHT1 proteins to visualize localization

  • Quantify plasma membrane vs. internal signal intensity

  • The cnih5 mutant typically shows reduced PM targeting efficiency of AtPHT1;1 in root hair and epidermis within the transition/elongation zone

What proteomic approaches are recommended for identifying AtCNIH5-dependent trafficking proteins?

For comprehensive identification of AtCNIH5-dependent trafficking proteins, a multi-stage proteomic approach is recommended:

• Sample preparation using specialized solubilization:

  • Apply UV-cleavable 4-hexylphenylazosulfonate (Azo)-solubilized microsomal protein extraction

  • This method is particularly effective for membrane proteins

  • Prepare samples from both wild-type and cnih5 mutant plants

  • Use Pi-limited conditions to maximize AtCNIH5 expression

• Quantitative proteomics using iTRAQ labeling:

  • Label peptides with isobaric tags for relative quantification

  • Combine samples for simultaneous analysis

  • Perform LC-MS/MS for protein identification and quantification

  • This approach identified 4,317 proteins in Pi-limited Arabidopsis roots, with 372 upregulated and 106 downregulated in cnih5

• Data analysis workflow:

  • Filter for significant changes (typically p<0.05, fold change >1.5)

  • Perform Gene Ontology (GO) enrichment analysis

  • Categorize by cellular component, molecular function, and biological process

  • Identify membrane proteins and trafficking components

  • Validate key candidates using independent methods

• Validation of candidates:

  • Confirm protein-protein interactions using methods described in FAQ 3.2

  • Verify localization changes using fluorescent protein fusions

  • Assess functional relevance through genetic complementation

How can AtCNIH5 be utilized to enhance phosphate use efficiency in plants?

AtCNIH5 offers several potential strategies for enhancing phosphate use efficiency in plants:

• Overexpression approaches:

  • Constitutive overexpression of AtCNIH5 enhances plant growth under both Pi repletion and limitation

  • Tissue-specific expression in roots may provide more targeted benefits

  • Create chimeric proteins with enhanced cargo binding capabilities

  • Research has shown that increasing in-situ AtCNIH5 expression/activity boosts plant growth under Pi repletion and limitation

• Enhancing PHT1 trafficking efficiency:

  • Co-express AtCNIH5 with high-affinity PHT1 transporters

  • This combination increases plasma membrane targeting of PHT1s

  • Results in more efficient Pi uptake from soil

  • Potentially reduces fertilizer requirements

• Engineering AtCNIH5 variants:

  • Modify cargo selectivity through targeted mutations

  • Enhance protein stability under various stress conditions

  • Optimize subcellular localization for maximal efficiency

  • The C-terminal acidic residue is not required for interaction with AtPHT1;1, suggesting flexibility in engineering

• Crop-specific optimization:

  • Identify and characterize CNIH5 orthologs in crop species

  • Compare cargo selection mechanisms across species

  • Develop crop-specific enhancement strategies

  • Consider tissue-specific promoters to avoid unintended consequences

What is the role of AtCNIH5 in the broader context of plant stress responses?

AtCNIH5 functions within a complex network of plant stress responses:

• Integration with Pi starvation response (PSR):

  • AtCNIH5 is upregulated specifically during Pi limitation

  • It facilitates the ER export of PHT1 transporters to enhance Pi acquisition

  • Acts as a low Pi-responsive hub that controls ER export of specific membrane cargoes

  • Interplays with AtPHF1 in a coordinated response to Pi limitation

• Connections to lipid metabolism:

  • AtCNIH5 facilitates trafficking of enzymes involved in very long-chain fatty acid biosynthesis

  • These lipids are components of extracellular aliphatic compounds

  • Changes in membrane lipid composition are part of Pi starvation responses

  • Suggests a role in membrane remodeling during stress adaptation

• Cell wall modification:

  • AtCNIH5 affects enzymes involved in nucleotide sugar biosynthesis for cell walls

  • Cell wall composition changes are common adaptations to various stresses

  • May contribute to altered root architecture during Pi limitation

  • Points to broader roles beyond direct Pi transport

• Potential crosstalk with other stress pathways:

  • The trafficking mechanism may be relevant to multiple abiotic stresses

  • Preliminary data suggests connections to drought and salt stress responses

  • Transporters like AtDTX21 and AtDTX35 (AtCNIH5 cargoes) are implicated in broader stress responses

  • Represents a central node in cellular adaptation to suboptimal conditions

What are the unresolved questions about AtCNIH5 structure-function relationships?

Several key questions remain regarding AtCNIH5 structure-function relationships:

• Cargo binding domain identification:

  • The precise domains responsible for cargo recognition remain undefined

  • Unlike other CNIHs, AtCNIH5's C-terminal acidic residue is not required for PHT1;1 interaction

  • Structural studies (X-ray crystallography or cryo-EM) are needed to elucidate binding interfaces

  • Understanding these domains could enable rational protein engineering

• Trafficking pathway regulation:

  • How is AtCNIH5 itself transported to ER exit sites?

  • What post-translational modifications regulate its activity?

  • How is degradation of AtCNIH5 controlled during Pi resupply?

  • Identifying regulatory partners would provide insights into dynamic control

• Selective cargo recognition mechanisms:

  • What determines the specificity of AtCNIH5 for different cargo proteins?

  • Are there common structural motifs in AtCNIH5-dependent cargoes?

  • How does AtCNIH5 discriminate between different PHT1 family members?

  • Comparative analysis with other plant CNIHs could identify unique features

• Interaction with COPII machinery:

  • The precise mechanisms of AtCNIH5 interaction with COPII components need clarification

  • How does the "IFRTL"-like motif in AtCNIH5 function in plant cells?

  • Are there plant-specific adaptations in the COPII-dependent export process?

  • Addressing these questions would illuminate fundamental aspects of plant membrane trafficking

How can high-throughput techniques advance our understanding of AtCNIH5 function?

Advanced high-throughput techniques offer promising approaches to expand our understanding of AtCNIH5 function:

• Proximity-based proteomics:

  • BioID or TurboID fusion proteins to identify proximal interacting partners

  • APEX2-based proximity labeling for temporal dynamics of interactions

  • These methods can identify transient interactions missed by traditional approaches

  • Would help map the complete interaction network of AtCNIH5 in vivo

• Single-cell transcriptomics and proteomics:

  • Analyze cell type-specific responses to Pi limitation

  • Identify differential regulation of AtCNIH5 across root cell types

  • Correlate with downstream effectors in specific cellular contexts

  • Would address the cell type-dependent function of AtCNIH5

• Cryo-electron tomography:

  • Visualize 3D organization of ER exit sites containing AtCNIH5

  • Map spatial relationships between AtCNIH5, cargo proteins, and COPII components

  • Track vesicle formation and trafficking in native cellular environments

  • Would provide unprecedented structural insights at the subcellular level

• CRISPR-based screens:

  • Genome-wide screens for modifiers of AtCNIH5 function

  • Identify new components of the trafficking pathway

  • Discover genetic interactions through combinatorial mutagenesis

  • Would place AtCNIH5 in broader cellular networks

What are the evolutionary implications of AtCNIH5's specialized function in phosphate homeostasis?

The evolutionary aspects of AtCNIH5's specialized function raise intriguing questions:

• Comparative genomics across plant lineages:

  • CORNICHON proteins exist across eukaryotes but show functional diversification

  • AtCNIH5's specialized role in Pi homeostasis may represent a plant-specific adaptation

  • Comparative analysis across diverse plant species could reveal evolutionary trajectories

  • Special interest in species adapted to Pi-limited environments

• Functional divergence within the CNIH family:

  • Why does Arabidopsis maintain five CNIH genes with different functions?

  • How did AtCNIH5 acquire specificity for PHT1 transporters?

  • What selective pressures drove this specialization?

  • Understanding this divergence could illuminate adaptations to nutrient limitation

• Co-evolution with cargo proteins:

  • Have PHT1 transporters co-evolved with AtCNIH5?

  • Are there correlated changes in interacting domains?

  • How does this relationship compare across species with different Pi acquisition strategies?

  • Molecular evolution studies could identify signatures of selection

• Integration with Pi sensing and signaling pathways:

  • How did AtCNIH5 become integrated into Pi starvation responses?

  • What transcriptional and post-transcriptional regulatory mechanisms evolved?

  • Is this integration conserved across plant lineages?

  • Understanding this integration would provide insights into the evolution of nutrient homeostasis networks