Recombinant Arabidopsis thaliana ALA-interacting subunit 5 (ALIS5)

What is ALIS5 and what is its primary function in Arabidopsis thaliana?

ALIS5 (ALA-interacting subunit 5) functions as a beta-subunit that interacts with P4-type ATPases (P4 ATPases) in Arabidopsis thaliana. The protein forms complexes with ALA-family proteins (particularly ALA2 and ALA3) that act as lipid flippases, catalyzing phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes. These complexes are critical components of the cellular machinery required for proper secretory processes during plant development. The ALIS5 subunit appears essential for the functional activity of its associated ALA proteins in the transport process .

How does ALIS5 differ from other ALA-interacting subunits in Arabidopsis?

ALIS5 is part of a family of ALA-interacting subunits (ALIS1-5) in Arabidopsis thaliana. While ALIS proteins share structural similarities, they demonstrate different expression patterns and may interact preferentially with specific ALA proteins. For example, ALIS1 shows strong affinity for ALA3 and localizes to Golgi-like structures, particularly in root peripheral columella cells. ALIS5, by comparison, has been extensively studied for its interaction with ALA2, forming a complex with specific phospholipid recognition capabilities. The differentiation in expression patterns suggests tissue-specific roles for different ALA-ALIS complexes in plant development and membrane homeostasis .

What cellular compartment does ALIS5 primarily localize to?

ALIS5 has been found to localize to cellular membranes, particularly when complexed with ALA proteins. When interacting with ALA3, the complex localizes to the Golgi apparatus, which is critical for its role in secretory processes. This Golgi localization is significant for understanding how the ALA-ALIS complexes contribute to vesicle budding and membrane dynamics in plant cells. The localization pattern suggests ALIS5's involvement in the secretory pathway, particularly in processes related to polysaccharide transport and cell wall development .

How should researchers approach protein domain analysis of ALIS5 to understand its interaction with ALA proteins?

Researchers should employ multiple complementary approaches to analyze ALIS5 domains:

• Bioinformatic analysis: Use protein prediction tools to identify conserved domains, transmembrane regions, and potential interaction sites.

• Site-directed mutagenesis: Create targeted mutations in predicted interaction domains to assess their impact on ALA binding and complex formation.

• Co-immunoprecipitation with domain deletions: Generate truncated ALIS5 variants to map the minimal regions required for ALA interaction.

• Crosslinking studies: Utilize chemical crosslinkers to capture and identify direct contact points between ALIS5 and ALA proteins.

• Fluorescence resonance energy transfer (FRET): Tag domains with fluorescent proteins to visualize interaction dynamics in living cells.

These approaches should be performed in parallel with functional assays measuring lipid flippase activity to correlate structural features with biological function .

What are the optimal expression systems for producing recombinant ALIS5 protein?

• Protein folding requirements: As a membrane-associated protein, ALIS5 may require post-translational modifications or specific membrane environments for proper folding.

• Co-expression needs: For functional studies, co-expression with appropriate ALA partners (such as ALA2 or ALA3) may be necessary.

• Scale requirements: Small-scale analytical studies may use E. coli, while larger functional studies might benefit from eukaryotic expression systems.

• Tag selection: While His-tags have been effectively used, other affinity tags (FLAG, GST) may be suitable depending on the experimental goals and downstream applications .

What purification strategies yield the highest purity and activity for recombinant ALIS5?

To achieve optimal purity and activity of recombinant ALIS5, researchers should implement a multi-step purification strategy:

• Initial capture: Utilize affinity chromatography based on the fusion tag (e.g., Ni-NTA for His-tagged ALIS5).

• Membrane protein considerations: Include appropriate detergents during extraction and purification to maintain protein stability and prevent aggregation.

• Additional purification steps: Follow affinity purification with size exclusion chromatography to separate monomeric ALIS5 from aggregates and further remove impurities.

• Complex formation: For functional studies, consider co-purification with ALA partners to maintain the native complex structure.

• Quality control: Assess protein purity using SDS-PAGE and Western blotting with specific antibodies against ALIS5 or the affinity tag.

• Activity preservation: Store purified protein in buffer containing 50% glycerol at -20°C or -80°C, with working aliquots kept at 4°C for up to one week to prevent repeated freeze-thaw cycles .

How should researchers approach reconstitution of the ALA2-ALIS5 complex for biochemical assays?

For successful reconstitution of the ALA2-ALIS5 complex for biochemical assays, researchers should follow these methodological considerations:

• Co-expression strategy: Express both ALA2 and ALIS5 proteins simultaneously in the same system to promote natural complex formation during synthesis.

• Sequential purification: Utilize tandem affinity purification with different tags on each protein (e.g., FLAG-tagged ALIS5 and His-tagged ALA2) to ensure isolation of the complete complex.

• Membrane environment: Reconstitute the purified complex into liposomes with a phospholipid composition mimicking the native membrane environment, particularly including phosphatidylserine.

• Functional verification: Assess complex formation using analytical techniques such as blue native PAGE, gel filtration chromatography, or co-immunoprecipitation with antibodies against both components.

• Activity testing: Measure ATP hydrolytic activity using established enzymatic assays, with particular attention to phosphatidylserine-stimulated activity as a marker of properly formed complex .

How can researchers effectively measure the lipid flippase activity of ALA-ALIS5 complexes?

To effectively measure lipid flippase activity of ALA-ALIS5 complexes, researchers should consider these methodological approaches:

• Fluorescent lipid analogs: Utilize fluorescently labeled phospholipids (particularly phosphatidylserine) and measure their translocation across the membrane bilayer using stopped-flow fluorescence spectroscopy.

• ATP hydrolysis assays: Measure ATP consumption rates in the presence of different phospholipid substrates, as the lipid flippase activity is coupled to ATP hydrolysis. This approach revealed that the ALA2-ALIS5 complex shows highly specific stimulation by phosphatidylserine.

• Reconstituted proteoliposome systems: Incorporate purified ALA-ALIS5 complexes into artificial liposomes with defined lipid compositions to control the experimental environment.

• NBD-labeled lipid assays: Use lipids labeled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) to track translocation, with subsequent dithionite quenching to distinguish between inner and outer leaflet distributions.

• Comparative substrate analysis: Test various lipid head groups and acyl chain compositions systematically to determine substrate specificity profiles. Research has shown that the lipid head group is the key structural element for substrate recognition by the P4 ATPase .

What approaches can determine the specific regions of ALIS5 responsible for ALA protein interaction?

To identify specific regions of ALIS5 responsible for ALA protein interaction, researchers should employ these complementary approaches:

• Truncation analysis: Generate systematic N-terminal and C-terminal truncations of ALIS5 to map the minimal interaction domain required for ALA binding.

• Yeast two-hybrid screening: Use fragmented ALIS5 constructs as bait against ALA prey constructs to identify interacting domains in a cellular context.

• Pull-down assays: Express recombinant fragments of ALIS5 with affinity tags and assess their ability to capture ALA proteins from cell lysates.

• Co-expression studies: Express various ALIS5 mutants alongside ALA2 or ALA3 in heterologous systems (such as yeast) to assess functional complementation, as demonstrated in studies showing that ALA3 function requires interaction with members of the ALIS family.

• Structural analysis: Where possible, employ X-ray crystallography or cryo-electron microscopy of the complex to visualize interaction interfaces at atomic resolution.

• Crosslinking mass spectrometry: Use chemical crosslinkers followed by mass spectrometric analysis to identify residues in close proximity between ALIS5 and ALA proteins .

How does the substrate specificity of the ALA2-ALIS5 complex compare with other P4-ATPase complexes?

The ALA2-ALIS5 complex demonstrates highly specific substrate recognition characteristics compared to other P4-ATPase complexes:

• Head group specificity: The ALA2-ALIS5 complex shows remarkably specific stimulation by phosphatidylserine, with the lipid head group serving as the key structural element for substrate recognition. Even small changes in the stereochemistry or functional groups of the phosphatidylserine head group significantly affect enzymatic activity.

• Acyl chain flexibility: Unlike some P4-ATPases that show strict acyl chain requirements, the ALA2-ALIS5 complex shows only minor effects from alterations in acyl chain length and composition, demonstrating head group primacy in substrate selection.

• Mono- vs. di-acyl tolerance: The ALA2-ALIS5 complex is stimulated by both mono- and di-acyl phosphatidylserines, suggesting a recognition mechanism primarily focused on the head group rather than the hydrophobic portions of the substrate.

• Comparative specificity profiles: Other P4-ATPases may recognize different phospholipids (phosphatidylcholine, phosphatidylethanolamine) or show broader substrate ranges, making the high phosphatidylserine specificity of ALA2-ALIS5 a distinctive feature for comparative studies of recognition mechanisms .

What are the methodological considerations for studying ALIS5 in the context of plant membrane dynamics?

When studying ALIS5 in the context of plant membrane dynamics, researchers should consider these methodological approaches:

• Subcellular localization studies: Employ fluorescent protein fusions and confocal microscopy to track ALIS5 localization in living plant cells, with particular attention to co-localization with organelle markers (especially Golgi apparatus markers).

• Membrane fractionation: Use differential centrifugation and density gradient separation to isolate specific membrane compartments containing ALIS5-ALA complexes.

• Lipid composition analysis: Combine lipidomics approaches with ALIS5 localization data to correlate membrane lipid asymmetry with ALIS5-ALA complex presence.

• Genetic manipulation: Generate knockout/knockdown plants or use CRISPR-Cas9 genome editing to create ALIS5 mutants for studying resultant changes in membrane organization and plant development.

• Vesicle trafficking assays: Employ fluorescent membrane tracers to assess how ALIS5 manipulation affects vesicle formation, budding, and fusion dynamics, particularly in the Golgi apparatus where ALA3-ALIS complexes are known to localize.

• Electron microscopy: Use immunogold labeling to visualize ALIS5 distribution at ultrastructural resolution, particularly focusing on membrane deformation sites .

How can researchers generate and validate ALIS5 mutants to assess functional domains?

To generate and validate ALIS5 mutants for functional domain assessment, researchers should follow these methodological guidelines:

• Rational design approach: Use sequence alignment with other ALIS proteins, structural predictions, and evolutionary conservation analysis to identify candidate residues for mutagenesis.

• Mutagenesis strategy selection:

  • Site-directed mutagenesis for changing specific amino acids

  • Deletion mutagenesis for removing entire domains

  • Chimeric constructs swapping domains between different ALIS proteins

• Expression system considerations: Express mutants in heterologous systems like yeast that lack endogenous P4-ATPases to avoid background complications, similar to the yeast complementation experiments described where ALA3 function required interaction with ALIS family members.

• Functional validation assays:

  • Co-immunoprecipitation to assess ALA protein binding

  • Subcellular localization to verify proper targeting

  • Lipid translocation assays to measure flippase activity

  • ATP hydrolysis measurements to quantify enzymatic function

• Complementation testing: Introduce mutant ALIS5 variants into ALIS5-deficient plants to assess restoration of normal phenotypes and membrane functions.

• Controls: Include non-mutated wild-type ALIS5 and known non-functional mutants as positive and negative controls in all experiments .

What are common pitfalls when working with recombinant ALIS5 and how can they be addressed?

When working with recombinant ALIS5, researchers frequently encounter several challenges that can be addressed with specific strategies:

• Low expression yields:

  • Optimize codon usage for the expression host

  • Adjust induction conditions (temperature, inducer concentration, time)

  • Consider fusion partners that enhance solubility

  • Test different promoter strengths and expression hosts

• Protein aggregation:

  • Include appropriate detergents during extraction and purification

  • Optimize buffer conditions (pH, salt concentration, glycerol addition)

  • Consider mild solubilization conditions to maintain native structure

  • Avoid repeated freeze-thaw cycles by storing in working aliquots at 4°C

• Loss of interaction with ALA partners:

  • Co-express ALIS5 with its ALA partner

  • Ensure membrane environment mimics native conditions

  • Preserve critical post-translational modifications

• Functional activity loss:

  • Verify protein folding using circular dichroism

  • Include lipid components that stabilize the complex

  • Maintain gentle handling during purification steps

  • Store with 50% glycerol as specified for commercial preparations

• Degradation during purification:

  • Include protease inhibitors in all buffers

  • Minimize purification time and keep samples cold

  • Consider epitope tag positioning to avoid interfering with function .

How should researchers interpret contradictory results in ALIS5 functional studies?

When faced with contradictory results in ALIS5 functional studies, researchers should employ these analytical and methodological approaches:

• Context-dependent function analysis:

  • Compare experimental conditions between contradictory studies (expression systems, purification methods, assay conditions)

  • Assess differences in complex formation (ALA2-ALIS5 vs. ALA3-ALIS5)

  • Consider tissue-specific or developmental context differences

• Technical validation:

  • Confirm protein identity using mass spectrometry

  • Verify complex formation using multiple independent methods

  • Assess protein quality through activity assays against established standards

• Substrate considerations:

  • Evaluate differences in lipid compositions used across studies

  • Compare mono-acyl versus di-acyl phospholipid substrates

  • Examine head group specificity under different experimental conditions

• Comparative replication:

  • Replicate key experiments from contradictory studies side-by-side

  • Systematically vary one condition at a time to identify the source of discrepancy

  • Include appropriate positive and negative controls

• Biological relevance assessment:

  • Connect in vitro findings with in vivo phenotypes

  • Consider the physiological context of observed differences

  • Evaluate whether contradictions reflect genuine biological complexity or technical artifacts .

What unexplored aspects of ALIS5 function present promising research opportunities?

Several unexplored aspects of ALIS5 function present promising research opportunities:

• Regulatory mechanisms:

  • Investigation of post-translational modifications that regulate ALIS5-ALA interactions

  • Exploration of potential dynamic assembly/disassembly of complexes in response to cellular conditions

  • Analysis of transcriptional and translational control mechanisms in different tissues

• Structural biology:

  • Determination of the complete three-dimensional structure of ALIS5 alone and in complex with ALA partners

  • Elucidation of conformational changes during the catalytic cycle

  • Mapping of lipid binding sites and their structural dynamics

• Systems biology:

  • Network analysis of ALIS5 interactions beyond ALA proteins

  • Investigation of ALIS5 role in broader membrane homeostasis pathways

  • Comparative analysis across plant species to understand evolutionary conservation and divergence

• Applied biotechnology:

  • Exploration of ALIS5 manipulation for enhanced stress tolerance in plants

  • Development of ALIS5-based biosensors for lipid dynamics

  • Engineering modified ALIS5 variants with novel substrate specificities

• Cross-talk with other membrane processes:

  • Investigation of ALIS5 roles in specialized secretory pathways

  • Analysis of interactions with vesicle trafficking machinery

  • Exploration of potential roles in plant-specific membrane dynamics during development .

How might advanced imaging techniques enhance our understanding of ALIS5 dynamics in living cells?

Advanced imaging techniques offer powerful approaches to enhance our understanding of ALIS5 dynamics in living cells:

• Super-resolution microscopy:

  • Stimulated emission depletion (STED) microscopy to visualize ALIS5 localization beyond the diffraction limit

  • Single-molecule localization microscopy (PALM/STORM) to track individual ALIS5 molecules in membranes

  • Structured illumination microscopy (SIM) to observe ALIS5 distribution within organelle subdomains

• Live-cell dynamics:

  • Fluorescence recovery after photobleaching (FRAP) to measure ALIS5 mobility within membranes

  • Fluorescence correlation spectroscopy (FCS) to analyze diffusion properties and complex formation

  • Single-particle tracking to follow ALIS5-containing vesicles during trafficking events

• Protein-protein interactions:

  • Förster resonance energy transfer (FRET) imaging to visualize ALIS5-ALA interactions in real-time

  • Bimolecular fluorescence complementation (BiFC) to map interaction domains in living cells

  • Proximity ligation assays to detect and quantify protein interactions with high sensitivity

• Functional imaging:

  • Genetically encoded lipid sensors to correlate ALIS5 activity with membrane lipid asymmetry

  • Optogenetic tools to manipulate ALIS5 function with spatiotemporal precision

  • Correlative light and electron microscopy to connect ALIS5 localization with membrane ultrastructure

• Multi-dimensional imaging:

  • 4D imaging (3D + time) to track ALIS5 dynamics during developmental processes

  • Multi-color imaging to simultaneously visualize ALIS5, ALA partners, and membrane markers

  • Light sheet microscopy for long-term, low-phototoxicity observation of ALIS5 dynamics .

How can computational modeling enhance our understanding of ALIS5-ALA complex function?

Computational modeling approaches can significantly enhance our understanding of ALIS5-ALA complex function through several specific applications: