Recombinant Arabidopsis thaliana ALA-interacting subunit 3 (ALIS3)

What is Arabidopsis thaliana ALIS3 and how does it function in plant cellular processes?

ALIS3 (ALA-Interacting Subunit 3) is a member of the ALIS protein family in Arabidopsis thaliana, which includes five members (ALIS1 to ALIS5). These proteins function as β-subunits for P4-ATPases, particularly ALA3. The ALIS proteins are homologs of the yeast Cdc50p protein family. In Arabidopsis, ALIS proteins interact with ALA P4-ATPases to form complexes that are essential for lipid translocation across membranes. The ALA-ALIS protein complexes are involved in generating lipid asymmetry between membrane leaflets and in inducing membrane curvature, which is critical for vesicle budding and secretory processes during plant development .

How does ALIS3 relate to other members of the ALIS family?

ALIS3 is one of five members (ALIS1-ALIS5) of the ALA-Interacting Subunit family in Arabidopsis. While all ALIS proteins can potentially interact with ALA3, research has shown particularly strong affinity between ALA3 and ALIS1, which can be co-purified in a detergent-resistant protein complex. The ALIS family members share similar functions as β-subunits of P4-ATPases, but they may have different expression patterns and specific interactions with various ALA proteins throughout the plant. The comparative analysis of ALIS family members provides insights into the specialized roles of these protein interactions in different tissues and developmental stages .

What are the optimal conditions for storing and reconstituting recombinant ALIS3 protein?

Recombinant ALIS3 protein is typically supplied as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE. For optimal storage and reconstitution:

The addition of glycerol is crucial for long-term storage as it prevents protein degradation during freeze-thaw cycles. Reconstituted protein should be aliquoted to avoid repeated freeze-thaw cycles which can compromise protein integrity and biological activity .

How can researchers verify the functionality of recombinant ALIS3 after reconstitution?

To verify ALIS3 functionality after reconstitution, researchers should assess both the protein's structural integrity and its biological activity:

• Structural Integrity Assessment:

  • SDS-PAGE analysis to confirm protein size and purity

  • Western blotting with anti-His antibodies to verify the presence of the His-tag

  • Circular dichroism spectroscopy to evaluate secondary structure integrity

• Functional Verification:

  • Protein-protein interaction assays with ALA3 using co-immunoprecipitation or pull-down assays

  • Lipid translocation assays in artificial membrane systems

  • Complementation assays in yeast mutants lacking endogenous Cdc50p proteins

The most definitive test is to demonstrate that the recombinant ALIS3 can interact with ALA3 and restore functionality in systems where either protein has been knocked out. This can be achieved through yeast complementation experiments similar to those performed with ALA3 and ALIS1, where interaction between these proteins was shown to be essential for ALA3 function .

What expression systems beyond E. coli have been successful for producing functional ALIS3?

While E. coli is commonly used for recombinant ALIS3 production, other expression systems may provide advantages for obtaining properly folded and post-translationally modified protein:

Research has demonstrated successful expression of functional ALIS proteins in yeast systems, particularly for interaction studies with ALA proteins. When ALA3 was expressed in a yeast strain (Δdrs2 Δdnf1 Δdnf2) deficient in P4-ATPases, the co-expression with ALIS proteins was found to be essential for ALA3 function, suggesting that yeast can produce functional ALIS proteins that properly interact with their ALA partners .

What experimental approaches are most effective for studying ALIS3-ALA interactions?

Several experimental approaches have proven effective for studying ALIS3-ALA interactions:

• Yeast Complementation Assays:

  • Expression of ALA3 and ALIS proteins in P4-ATPase-deficient yeast strains

  • Assessment of phenotype restoration (e.g., cold sensitivity in Δdrs2 Δdnf1 Δdnf2 mutants)

  • This approach demonstrated that interaction with ALIS family members is a strict requirement for ALA3 function

• Co-purification Techniques:

  • Detergent-resistant protein complex isolation

  • Affinity purification using tags on either protein

  • These techniques have shown that ALIS1 and ALA3 interact directly and can be co-purified

• Localization Studies:

  • Fluorescent protein fusions (e.g., GFP:ALA3)

  • Co-localization experiments in plant cells

  • These have confirmed that both ALA3 and ALIS1 localize to Golgi-like structures in planta

• Functional Assays:

  • Lipid translocation assays

  • Vesicle budding experiments

  • These approaches connect the physical interaction to biological function

Studies have shown that ALIS1 and ALA3 show particularly strong affinity for each other and form a functional complex essential for secretory processes in peripheral columella cells at the root tip .

How does the interaction between ALIS3 and ALA3 affect subcellular localization and function?

The interaction between ALIS proteins and ALA3 is critical for proper subcellular localization and function:

• Localization Effects:

  • In planta, both ALA3 and ALIS1 localize to Golgi-like structures

  • Proper ALA3 trafficking to the Golgi apparatus likely depends on interaction with ALIS proteins

  • GFP:ALA3 fusion protein localizes specifically to the Golgi apparatus and remains functional

• Functional Consequences:

  • The ALIS-ALA3 interaction is required for lipid translocation activity

  • This protein complex forms an essential part of the Golgi machinery involved in secretory processes

  • In the absence of interaction with ALIS proteins, ALA3 likely fails to reach its proper destination or cannot function properly

• Developmental Impact:

  • The ALA3/ALIS protein complex is particularly important in secretory processes in peripheral columella cells at the root tip

  • Disruption of this interaction leads to impaired vesicle budding from the trans-Golgi

  • This results in failure of the root cap to release border cells and impaired secretion of molecules required for efficient root interaction with the environment

The localization of ALA3 to the Golgi apparatus and its function in vesicle budding processes are dependent on interaction with ALIS proteins, highlighting the importance of this protein-protein interaction for proper cellular function .

What phenotypes are associated with ALIS3 mutations in Arabidopsis, and how do they compare to ALA3 mutant phenotypes?

The ala3 mutants show severe defects in vesicle production at secreting peripheral columella cells of the root tip. Because ALIS proteins are required for ALA3 function, mutations in ALIS genes might be expected to produce similar phenotypes, though potentially with different severity or tissue specificity depending on the redundancy and expression patterns of the different ALIS family members .

How can complementation assays with recombinant ALIS3 be designed to verify gene function in mutant backgrounds?

Designing effective complementation assays with recombinant ALIS3 involves several critical steps:

• Mutant Selection:

  • Identify and characterize alis3 knockout or knockdown mutants

  • Consider double or triple mutants of multiple ALIS genes to address functional redundancy

  • Select appropriate ALA3 mutant lines for testing ALIS3-ALA3 interaction effects

• Complementation Construct Design:

  • Create expression constructs with native promoters for most authentic expression

  • Include fluorescent tags for localization studies while ensuring they don't interfere with function

  • Develop inducible expression systems for temporal control of complementation

• Transformation and Selection:

  • Transform mutant plants with ALIS3 expression constructs

  • Select multiple independent transgenic lines with varying expression levels

  • Verify transgene expression through RT-PCR and protein detection methods

• Phenotypic Analysis:

  • Evaluate restoration of wild-type morphology, particularly in root and shoot development

  • Examine cellular phenotypes such as vesicle formation in the trans-Golgi

  • Assess border cell release from the root cap

  • Analyze secretory processes in peripheral columella cells

Similar to the GFP:ALA3 fusion protein, which was shown to be functional by its ability to complement the ala3-1 mutant phenotype, recombinant ALIS3 constructs can be tested for their ability to restore normal development and cellular functions in appropriate mutant backgrounds .

What techniques are most effective for analyzing ALIS3 expression patterns in different plant tissues?

Several complementary techniques can effectively analyze ALIS3 expression patterns:

• Promoter-Reporter Fusions:

  • Clone the ALIS3 promoter region upstream of reporter genes (GUS, GFP)

  • Transform plants and analyze reporter expression in different tissues

  • This approach can reveal the spatial and temporal regulation of ALIS3 expression

• RT-qPCR Analysis:

  • Design specific primers for ALIS3 to distinguish it from other ALIS family members

  • Collect RNA from different tissues and developmental stages

  • Quantify relative expression levels across tissues

• RNA-Seq Analysis:

  • Perform transcriptome analysis on different tissues

  • Analyze ALIS3 expression patterns in publicly available RNA-Seq datasets

  • Compare expression with other ALIS family members and ALA genes

• In Situ Hybridization:

  • Design specific probes for ALIS3 mRNA

  • Perform in situ hybridization on tissue sections

  • Visualize expression at the cellular level within intact tissues

For comparative purposes, research on ALA3 showed that its promoter was active in the vascular tissue in cells surrounding the xylem and in the columella root cap. During lateral root formation, expression was first evident in columella root cap initials and later appeared in all cells of the columella root cap. Similar approaches could be applied to study ALIS3 expression patterns .

What subcellular localization methods best resolve ALIS3 distribution in plant cells?

For optimal resolution of ALIS3 subcellular localization, several techniques can be employed:

• Fluorescent Protein Fusions:

  • Create N- or C-terminal fusions of ALIS3 with fluorescent proteins (GFP, mCherry)

  • Express in plant cells under native or suitable promoters

  • Verify functionality of fusion proteins through complementation assays

• Confocal Microscopy Techniques:

  • Standard confocal microscopy for basic localization

  • Super-resolution microscopy (STED, PALM, STORM) for detailed subcellular distribution

  • Live-cell imaging to track dynamic localization changes

• Co-localization Studies:

  • Use established organelle markers (e.g., Golgi, ER, plasma membrane markers)

  • Perform dual-color imaging with ALIS3 fusions and organelle markers

  • Calculate co-localization coefficients for quantitative assessment

• Immunogold Electron Microscopy:

  • Develop specific antibodies against ALIS3 or use anti-tag antibodies

  • Perform immunogold labeling on fixed tissue sections

  • Visualize precise subcellular localization at ultrastructural level

Similar to ALA3, which was shown to localize to the Golgi apparatus using GFP:ALA3 fusion proteins, ALIS3 localization can be studied using fluorescent protein fusions. The functionality of such fusions should be confirmed through complementation assays, as was done for GFP:ALA3 .

How can ALIS3 research contribute to understanding lipid asymmetry and membrane dynamics in plants?

ALIS3 research provides important insights into lipid asymmetry and membrane dynamics:

• Lipid Translocation Mechanisms:

  • ALIS3-ALA complexes function as lipid translocases (P4-ATPases)

  • These proteins are implicated in generating lipid asymmetry between membrane leaflets

  • Studying ALIS3 interactions helps elucidate how plants maintain membrane asymmetry

• Membrane Curvature and Vesicle Formation:

  • P4-ATPases contribute to inducing membrane curvature

  • This is essential for vesicle budding from donor membranes

  • ALIS3 research helps understand how plant cells regulate this process

• Golgi Function and Secretory Pathways:

  • ALA3-ALIS complexes localize to the Golgi apparatus

  • They play critical roles in vesicle budding from the trans-Golgi

  • ALIS3 studies contribute to understanding plant-specific aspects of the secretory pathway

• Plant-Specific Membrane Biology:

  • P4-ATPases and their β-subunits may have plant-specific functions

  • Understanding these differences contributes to the broader field of comparative membrane biology

  • Research on ALIS3 can highlight plant-specific adaptations in membrane dynamics

The ALA3/ALIS protein complexes are essential components of the Golgi machinery involved in secretory processes. Detailed understanding of these interactions provides insights into fundamental aspects of plant cell biology, particularly regarding membrane organization and dynamics .

What are the implications of ALIS3-ALA3 interactions for agricultural applications and crop improvement?

The ALIS3-ALA3 interactions have several potential implications for agriculture:

• Root Development and Nutrient Uptake:

  • ALA3 mutations affect root development and border cell release

  • Border cells are involved in secretion of molecules required for efficient root interaction with the environment

  • Understanding ALIS3-ALA3 functions could lead to improved root systems and nutrient acquisition in crops

• Stress Response Optimization:

  • Membrane lipid composition and asymmetry play roles in stress responses

  • Modulating ALIS-ALA interactions might enhance plant resilience to environmental stresses

  • This could lead to crops with improved drought or salt tolerance

• Secretory Pathway Engineering:

  • The secretory pathway is critical for cell wall formation and extracellular matrix composition

  • Manipulating ALIS-ALA functions could potentially optimize cell wall properties

  • This has implications for biomass quality, plant architecture, and pathogen resistance

• Translational Research Potential:

  • Findings from Arabidopsis ALIS3-ALA3 research can be translated to crop species

  • Identifying and characterizing orthologs in crops provides targets for breeding or engineering

  • Arabidopsis continues to serve as a nexus for discovery, innovation, and application in both plant and human biology

The fundamental knowledge gained from studying ALIS3-ALA3 interactions in Arabidopsis can contribute to sustainable crop production and climate change mitigation strategies, as highlighted in the 4th Multinational Arabidopsis Steering Committee Roadmap for research through 2030 .

How do ALIS proteins in Arabidopsis compare to similar proteins in other plant species and model organisms?

ALIS proteins in Arabidopsis can be compared to similar proteins across species:

What evolutionary insights can be gained from studying ALIS3 sequence conservation across plant species?

Evolutionary analysis of ALIS3 provides several important insights:

• Functional Domain Conservation:

  • Highly conserved domains likely represent critical functional regions

  • Less conserved regions may indicate species-specific adaptations

  • Transmembrane domains and interaction interfaces with ALA proteins would show evolutionary constraints

• Selection Pressure Analysis:

  • Patterns of selection (purifying, neutral, or positive) across the sequence

  • Sites under positive selection may indicate adaptive evolution

  • Purifying selection suggests functional constraints

• Gene Duplication Patterns:

  • Timing of ALIS gene family expansion in plant evolution

  • Potential neofunctionalization or subfunctionalization after duplication

  • Correlation with expansion of interacting ALA gene family

• Structure-Function Relationships:

  • Conservation patterns can guide identification of critical residues

  • Comparative modeling based on conserved sequences

  • Prediction of interaction surfaces with ALA proteins

Studying the 349-amino acid sequence of ALIS3 across plant species would reveal how this protein has evolved in relation to its function as a β-subunit for P4-ATPases, potentially identifying key residues for protein-protein interactions and membrane association .

What are common challenges in working with recombinant ALIS3 and how can they be addressed?

Researchers working with recombinant ALIS3 may encounter several challenges:

A critical consideration is that ALIS3, as a membrane protein with multiple transmembrane domains, requires appropriate handling to maintain its native structure and function. The addition of 5-50% glycerol during storage helps maintain protein stability, and avoiding repeated freeze-thaw cycles is essential for preserving activity .

How can researchers troubleshoot phenotypic inconsistencies in ALIS3 functional studies?

When encountering phenotypic inconsistencies in ALIS3 functional studies, researchers should consider:

• Genetic Redundancy Assessment:

  • Test for functional compensation by other ALIS family members

  • Consider creating double or triple mutants of ALIS genes

  • Examine expression changes of other ALIS genes in alis3 mutants

• Expression Level Variation:

  • Quantify ALIS3 expression levels in different experimental conditions

  • Ensure consistent transgene expression in complementation studies

  • Consider using inducible promoters for controlled expression

• Environmental Influences:

  • Standardize growth conditions (light, temperature, humidity)

  • Test phenotypes under different environmental stresses

  • Control for developmental stage variations

• Methodological Consistency:

  • Standardize phenotyping protocols

  • Implement quantitative measurements rather than qualitative assessments

  • Use multiple independent methods to verify phenotypes

• Genetic Background Effects:

  • Backcross mutants to ensure clean genetic background

  • Use multiple independent mutant alleles or CRISPR/Cas9-generated mutations

  • Consider natural variation in different Arabidopsis ecotypes

A comprehensive dataset of genes with loss-of-function mutant phenotypes in Arabidopsis has highlighted the importance of confirming gene-to-phenotype associations through molecular complementation or multiple alleles. This approach is particularly important for ALIS genes where redundancy may mask phenotypes in single mutants .

What emerging technologies could advance our understanding of ALIS3 function in plant membrane dynamics?

Several emerging technologies hold promise for advancing ALIS3 research:

• CRISPR/Cas9 Genome Editing:

  • Precise editing of ALIS3 coding sequences

  • Creation of protein variants with altered function

  • Targeting of multiple ALIS genes simultaneously to overcome redundancy

• Advanced Microscopy Techniques:

  • Super-resolution microscopy for detailed localization

  • Single-molecule tracking to study ALIS3 dynamics in live cells

  • Correlative light and electron microscopy for structural context

• Cryo-Electron Microscopy:

  • Structural determination of ALIS3-ALA3 complexes

  • Visualization of conformational changes during lipid translocation

  • Insights into molecular mechanisms of protein-protein interactions

• Lipidomics and Membrane Biophysics:

  • Comprehensive analysis of lipid composition changes in alis3 mutants

  • Biophysical studies of membrane properties in the presence/absence of ALIS3

  • Artificial membrane systems to study ALIS3-mediated lipid translocation

• Single-Cell Transcriptomics and Proteomics:

  • Cell-type-specific analysis of ALIS3 expression and function

  • Identification of co-regulated genes and proteins

  • Understanding cellular heterogeneity in response to ALIS3 manipulation

These technologies align with the computational approaches emphasized in the 4th Multinational Arabidopsis Steering Committee Roadmap, which highlights the importance of advanced tools for understanding complex biological systems in plant research through 2030 .

What are the most promising research questions regarding ALIS3 that remain to be addressed?

Several key research questions about ALIS3 remain to be fully addressed:

• Specificity of ALIS-ALA Interactions:

  • What molecular determinants govern specific interactions between ALIS3 and various ALA proteins?

  • How do different ALIS-ALA combinations affect substrate specificity and cellular function?

  • What is the stoichiometry and structural basis of these interactions?

• Regulatory Mechanisms:

  • How is ALIS3 expression and activity regulated during development and stress responses?

  • What post-translational modifications affect ALIS3 function?

  • How do cellular signaling pathways modulate ALIS3-ALA complex activity?

• Functional Redundancy and Specialization:

  • What are the unique functions of ALIS3 compared to other ALIS family members?

  • How do plants coordinate the activities of multiple ALIS proteins?

  • What evolutionary pressures led to the expansion of the ALIS gene family in plants?

• Integration with Cellular Processes:

  • How does ALIS3 function coordinate with other aspects of membrane trafficking machinery?

  • What role does ALIS3 play in plant-specific cellular processes?

  • How do ALIS3-dependent processes contribute to plant responses to biotic and abiotic stresses?

• Translational Potential:

  • Can manipulation of ALIS3 function improve agronomically important traits?

  • How conserved are ALIS3 functions in crop species?

  • What are the applications of ALIS3 research for synthetic biology approaches?

These questions align with the research priorities outlined in the Arabidopsis research roadmap for 2030, which emphasizes fundamental research, translation to crops, and addressing challenges related to climate change and sustainable production .

What is the current state of knowledge regarding ALIS3 and what are the key research milestones achieved?

The current state of knowledge regarding ALIS3 can be summarized as follows:

• Identification and Characterization:

  • ALIS3 is one of five members of the ALIS family in Arabidopsis

  • It is a 349-amino acid protein with transmembrane domains

  • ALIS proteins are homologs of the yeast Cdc50p family

  • Recombinant ALIS3 can be produced in E. coli expression systems

• Functional Understanding:

  • ALIS proteins function as β-subunits for P4-ATPases (like ALA3)

  • These complexes are involved in lipid translocation across membranes

  • They contribute to generating lipid asymmetry and inducing membrane curvature

  • Interaction with ALIS proteins is required for ALA3 function

• Cellular Localization:

  • ALIS1 (similar to ALIS3) localizes to Golgi-like structures

  • The ALA3-ALIS protein complex is part of the Golgi machinery

  • This complex is involved in vesicle budding from the trans-Golgi

• Developmental Roles:

  • The ALA3-ALIS complex is important for secretory processes during plant development

  • It plays a critical role in peripheral columella cells at the root tip

  • Disruption affects border cell release from the root cap

The field has progressed from initial identification of ALIS proteins to understanding their interactions with ALA proteins and their roles in fundamental cellular processes related to membrane dynamics and vesicle formation .

How should researchers approach designing comprehensive studies of ALIS3 function in plant development and stress responses?

A comprehensive approach to studying ALIS3 function should include:

• Integrated Multi-omics Strategy:

  • Combine transcriptomics, proteomics, and lipidomics analyses

  • Study effects of ALIS3 manipulation across developmental stages and stress conditions

  • Integrate data using computational approaches and systems biology

• Genetic Analysis Framework:

  • Generate clean knockout mutants using CRISPR/Cas9

  • Create higher-order mutants with other ALIS genes

  • Develop tissue-specific and inducible expression systems

  • Perform detailed phenotypic analyses under various conditions

• Structural-Functional Studies:

  • Determine the structure of ALIS3 alone and in complex with ALA proteins

  • Create targeted mutations based on structural information

  • Test functional consequences of specific amino acid changes

• Cellular and Developmental Analysis:

  • Use advanced imaging to track ALIS3 dynamics in living cells

  • Study membrane properties and vesicle formation in wild-type and mutant backgrounds

  • Examine developmental processes with high temporal and spatial resolution

• Translational Research Components:

  • Identify and characterize ALIS3 orthologs in crop species

  • Test effects of ALIS3 modification on agronomically relevant traits

  • Develop applications based on fundamental knowledge of ALIS3 function