Recombinant Bovine Atlastin-1 (ATL1)

Basic Research Questions

• What is the basic structure and function of bovine Atlastin-1?

  Bovine Atlastin-1 (ATL1) is a dynamin-like GTPase of 558 amino acids that belongs to the dynamin superfamily. It contains a canonical large GTPase (G) domain, a middle domain, and two transmembrane segments near the C-terminus. The protein is primarily involved in endoplasmic reticulum (ER) membrane fusion. Structurally, ATL1 contains several key regions: the GTPase domain essential for GTP binding and hydrolysis, a middle three-helix bundle (3HB) domain, and transmembrane domains that anchor it to the ER membrane. The full-length bovine ATL1 sequence (1-558aa) includes critical regions for nucleotide-dependent dimerization, which facilitates membrane fusion activities .

• How is recombinant bovine ATL1 typically expressed and purified for research purposes?

  Recombinant bovine ATL1 is commonly expressed in E. coli expression systems using plasmid vectors that incorporate His-tags for purification. The typical protocol involves:

  • Cloning the full-length bovine ATL1 gene (1-558aa) into an expression vector with an N-terminal His-tag

  • Transforming the construct into E. coli expression hosts

  • Inducing protein expression under optimized conditions

  • Cell lysis followed by affinity chromatography using Ni-NTA columns

  • Further purification steps may include size exclusion chromatography

  • The purified protein is typically stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0

  • For long-term storage, the protein is often lyophilized or stored at -20°C/-80°C with 5-50% glycerol to maintain stability

  Protein purity is typically assessed by SDS-PAGE, with high-quality preparations achieving >90% purity.

• What assays can be used to verify the integrity and activity of recombinant bovine ATL1?

  Several assays can be employed to verify recombinant bovine ATL1 integrity and functionality:

  • GTPase activity assay: Measuring GTP hydrolysis rates using colorimetric or HPLC-based methods

  • Dimerization assays: Using size exclusion chromatography, analytical ultracentrifugation, or FRET-based approaches to assess nucleotide-dependent dimerization

  • Membrane fusion assays: In vitro liposome fusion assays using fluorescently labeled lipids

  • Western blotting: Using specific antibodies (such as 12149-1-AP or D2E6 Rabbit mAb) that cross-react with bovine ATL1 to confirm protein expression and integrity

  • Circular dichroism: To verify proper protein folding

  Activity assessment should include both nucleotide binding and hydrolysis, as mutations affecting these functions are known to impact ATL1 activity dramatically .

Advanced Research Questions

• How do different mutations in bovine ATL1 affect its function compared to human pathological variants?

  Research comparing bovine and human ATL1 mutations reveals important structural and functional insights:

  Mutations in human ATL1 associated with hereditary spastic paraplegia (HSP) type 3A have varying effects on protein function. Similar mutations introduced into bovine ATL1 can provide a comparative model for understanding these effects:

  Human Mutation	Bovine Equivalent	GTPase Activity	Dimerization	Fusion Activity	Notes
  R217Q	R192Q	Severely reduced	Defective	Defective	Complete loss of function in GTP hydrolysis
  R239C	R214C	Near normal	Normal	Slightly reduced	Common pathological mutation despite minor in vitro defects
  C375R	C350R	Unknown	Unknown	Unknown	Protein stability/folding issues observed
  M408T	M383T	Slightly reduced	Slightly reduced	Unknown	Modest impairment of function

  These comparative studies indicate that pathological mutations have a spectrum of effects on ATL1 function, from severe impairment (R217Q/R192Q) to subtle alterations (R239C/R214C). In vivo studies in Drosophila have shown that all pathological mutations reduce atlastin activity, but to different degrees of severity .

• What experimental approaches can be used to study the role of bovine ATL1 in ER morphogenesis?

  Several sophisticated approaches can be employed to study bovine ATL1's role in ER morphogenesis:

  • Liposome reconstitution systems: Purified recombinant bovine ATL1 can be reconstituted into liposomes with an ER-like lipid composition to study membrane fusion activities in vitro

  • Live-cell imaging with fluorescent markers: Co-expression of bovine ATL1 with ER markers (such as BiP-sfGFP-HDEL or ER-sfGFP-3) to visualize ER morphology changes in real-time

  • CRISPR/Cas9-mediated genome editing: Generation of specific mutations in the endogenous ATL1 gene to study their effects on ER structure under physiological conditions

  • Super-resolution microscopy: Techniques like STED or STORM microscopy can be used to visualize detailed ER structural changes induced by ATL1 variants

  • Electron microscopy: To examine ultrastructural changes in ER morphology at high resolution

  • Functional complementation assays: Testing whether bovine ATL1 can rescue ER morphology defects in cells depleted of endogenous atlastins

• How can I design experiments to assess the GTPase activity of recombinant bovine ATL1?

  Designing experiments to assess GTPase activity of recombinant bovine ATL1 requires precise methodology:

  Protocol outline for GTPase activity measurement:

  1. Protein preparation:

     • Purify recombinant bovine ATL1 to >90% purity

     • Use freshly prepared protein or carefully thawed aliquots to avoid freeze-thaw damage

     • Buffer composition: Tris/PBS-based buffer, pH 8.0 with 6% trehalose

  2. GTPase assay setup:

     • Reaction mixture: ATL1 (0.5-2 μM), GTP (100-500 μM), MgCl₂ (1-5 mM), buffer at pH 7.2-8.0

     • Include controls: no-protein control, GDP-loaded protein, and known GTPase-deficient mutant (R192Q equivalent)

     • Maintain temperature at 37°C for optimal enzymatic activity

  3. Activity measurement options:

     • Malachite green assay: Measures released inorganic phosphate

     • HPLC analysis: Monitors conversion of GTP to GDP

     • Coupled enzymatic assay: Using pyruvate kinase and lactate dehydrogenase to couple GTP hydrolysis to NADH oxidation

     • Radiolabeled GTP assay: For high sensitivity measurements

  4. Data analysis:

     • Calculate initial rates from linear portion of progress curves

     • Determine kinetic parameters (Km, Vmax, kcat) through Michaelis-Menten analysis

     • Compare activity of wild-type vs. mutant proteins under identical conditions

  For more accurate results, consider membrane reconstitution systems, as ATL1's GTPase activity can be enhanced by proper membrane association .

• What techniques can be used to study nucleotide-dependent dimerization of recombinant bovine ATL1?

  Nucleotide-dependent dimerization of bovine ATL1 can be studied using several complementary techniques:

  • Size Exclusion Chromatography (SEC): Monitor the shift in elution profile upon addition of different nucleotides (GDP, GTP, non-hydrolyzable GTP analogs). Crystal structures suggest GDP-bound dimers form, but solution studies indicate dimerization occurs primarily with GTP or transition state analogs .

  • Analytical Ultracentrifugation (AUC): Provides precise determination of molecular weight and oligomeric state in solution under different nucleotide conditions.

  • Small-Angle X-ray Scattering (SAXS): Analyzes the solution conformation of ATL1 dimers, providing insights into extended vs. compact conformations under different nucleotide states .

  • Förster Resonance Energy Transfer (FRET): Using fluorescently labeled ATL1 to monitor real-time dimerization kinetics.

  • Chemical Crosslinking coupled with Mass Spectrometry: Identifies specific residues involved in dimer interface formation.

  • Surface Plasmon Resonance (SPR): Measures binding kinetics and affinity between ATL1 monomers under different nucleotide conditions.

  When designing these experiments, researchers should consider that ATL1 dimerization in solution is primarily observed with GTP and transition state analogs, not with GDP, despite crystal structures showing GDP-bound dimers .

• How does the localization and trafficking of bovine ATL1 compare to human ATL1 in cellular models?

  Comparative analysis of bovine and human ATL1 trafficking provides important insights:

  • Subcellular localization: Both bovine and human ATL1 primarily localize to the endoplasmic reticulum (ER), particularly in regions of high membrane curvature. This can be visualized using fluorescently tagged proteins or immunofluorescence with specific antibodies like 12149-1-AP or D2E6 .

  • Expression patterns: Human ATL1 is predominantly expressed in the brain and spinal cord, particularly in neurons of the corticospinal tracts . Bovine ATL1 shows a similar tissue distribution, though some species-specific differences may exist.

  • Membrane dynamics: Both proteins participate in homotypic ER membrane fusion, but differences in fusion efficiency may be observed in heterologous expression systems.

  • Interaction with other proteins: Co-immunoprecipitation (CoIP) studies can reveal whether bovine ATL1 interacts with the same partner proteins as human ATL1 . Potential interacting partners include other ER-shaping proteins and components of the secretory pathway.

  • Response to mutations: Introducing equivalent mutations (like R192Q/R217Q or R214C/R239C) can reveal species-specific differences in how these proteins respond to pathological changes .

  For accurate comparison, parallel expression of bovine and human ATL1 constructs in the same cellular background (such as HeLa cells with endogenous atlastins depleted) is recommended to control for cell type-specific effects.

• What role does bovine ATL1 play in ER-phagy and how can this be experimentally assessed?

  Recent research indicates that atlastins, including ATL1, are key positive effectors of ER-phagy (selective autophagy of the ER) . To experimentally assess bovine ATL1's role in this process:

  1. Expression analysis: Determine which atlastin isoforms are dominantly expressed in bovine tissues of interest. Different cell types exhibit varying expression patterns of ATL1, ATL2, and ATL3 .

  2. Knockdown/knockout approaches: Use CRISPR-mediated gene editing or RNAi to deplete bovine ATL1 and assess effects on:

     • ER morphology using ER markers like BiP-sfGFP-HDEL

     • Autophagic flux using LC3 conversion assays

     • Accumulation of ER fragments using electron microscopy

     • ER stress markers like HSPA5, HSP90B1, and PDIA4

  3. ER-phagy assays: Employ quantitative assays such as the EATR (ER-phagy assay with tandem reporters) or CCER systems in bovine cell lines with ATL1 modification .

  4. Stress induction: Compare ER-phagy responses between wild-type and ATL1-depleted cells under stress conditions like nutrient starvation or ER stress inducers.

  5. Interaction studies: Investigate interactions between bovine ATL1 and known ER-phagy receptors like FAM134B, RTN3L, or CCPG1 using co-immunoprecipitation or proximity labeling approaches.

  6. Time-resolved interactome profiling: Apply temporal proteomics techniques to monitor how ATL1's interactions change during the induction of ER-phagy .

  When designing these experiments, consider that atlastin expression patterns vary significantly across different cell lines, with one or two dominantly expressed atlastins in each cell type .

• How can recombinant bovine ATL1 be used to study the mechanisms of membrane fusion in vitro?

  Recombinant bovine ATL1 can be employed in sophisticated in vitro systems to study membrane fusion mechanisms:

  Liposome Fusion Assay Protocol:

  1. Liposome preparation:

     • Prepare liposomes with an ER-like lipid composition (typically including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and cholesterol)

     • For fusion detection, incorporate fluorescent lipid pairs for FRET-based assays (e.g., NBD-PE and rhodamine-PE) in one population of liposomes

  2. Protein reconstitution:

     • Reconstitute purified recombinant bovine ATL1 into liposomes at protein:lipid ratios of 1:200 to 1:1000

     • Use detergent-mediated reconstitution followed by dialysis or detergent removal with Bio-Beads

     • Prepare control liposomes without protein and with GTPase-deficient mutants (R192Q equivalent)

  3. Fusion assay:

     • Mix labeled and unlabeled proteoliposomes in assay buffer containing GTP and MgCl₂

     • Monitor fusion by measuring FRET signal changes over time

     • Test effects of GTP concentration, temperature, and lipid composition on fusion efficiency

     • Include controls with GDP, non-hydrolyzable GTP analogs, or without nucleotides

  4. Advanced analysis:

     • Use dynamic light scattering to confirm liposome size and stability

     • Employ cryo-electron microscopy to visualize fusion intermediates

     • Consider single-vesicle fusion assays for more detailed kinetic analysis

  Recent research has demonstrated that human atlastins are sufficient to drive fusion of liposomes with appropriate lipid composition, and similar approaches can be applied to study bovine ATL1 . Compare results with those from human ATL1 to identify species-specific differences in fusion mechanisms.

• What experimental approaches can be used to study the involvement of bovine ATL1 in neurological disorders?

  To study the involvement of bovine ATL1 in neurological disorders, several experimental approaches can be employed:

  1. Comparative mutation analysis:

     • Introduce pathological mutations associated with human hereditary spastic paraplegia (HSP) into recombinant bovine ATL1 (such as R192Q, R214C, C350R, and M383T, corresponding to human R217Q, R239C, C375R, and M408T)

     • Assess effects on protein function using biochemical assays for GTPase activity, dimerization, and membrane fusion

  2. Primary neuron culture models:

     • Express wild-type or mutant bovine ATL1 in primary neurons

     • Analyze effects on:

       • ER morphology in neuronal cell bodies and axons

       • Axonal growth and guidance

       • Corticospinal tract development

       • Neuronal viability and function

  3. Interspecies comparison:

     • Compare bovine ATL1 structure and function with homologs from species known to develop spastic disorders, such as Bovine Spastic Syndrome (BSS), which may involve adenosine-A1-receptor homologs in striatal medium spiny neurons

     • Investigate potential cross-species conservation of pathogenic mechanisms

  4. Membrane trafficking studies:

     • Examine effects of ATL1 mutations on retrograde membrane trafficking using quantitative single-cell flow cytometry assays

     • Compare effects of wild-type and mutant bovine ATL1 on Golgi morphology and COPII formation

  5. Advanced imaging:

     • Use confocal microscopy to image BiP-sfGFP-HDEL-labeled ER in motor neurons expressing normal or mutant bovine ATL1

     • Employ live-cell imaging to track ER dynamics in neurons

  Understanding the neurological implications of bovine ATL1 can provide comparative insights into human neurological disorders like hereditary spastic paraplegia type 3A, where mutations in human ATL1 lead to progressive spasticity of the lower limbs .

Methodological Questions

• What are the best strategies for designing site-directed mutagenesis experiments on bovine ATL1?

  Optimal site-directed mutagenesis strategies for bovine ATL1 should consider:

  1. Target selection:

     • Focus on conserved residues between bovine and human ATL1

     • Key regions to target include:

       • GTPase domain: residues involved in GTP binding and hydrolysis

       • Middle domain: regions important for conformational changes

       • Transmembrane domains: residues affecting membrane insertion

     • Prioritize mutations corresponding to human pathological variants (R192Q, R214C, C350R, M383T)

  3. Validation strategies:

     • Confirm mutations by Sanger sequencing

     • Verify protein expression levels by Western blotting

     • Assess protein stability using thermal shift assays

     • Compare expression levels between wild-type and mutant proteins to control for this variable

  4. Functional characterization:

     • Test effects on GTPase activity, dimerization, and membrane fusion

     • Evaluate impacts on ER morphology in cellular models

     • Assess protein-protein interactions using co-immunoprecipitation

  5. Experimental controls:

     • Include both negative (non-functional mutant like R192Q) and positive (nearly wild-type function like R214C) controls

     • Use equivalent human mutations as comparative references

  This systematic approach allows for comprehensive characterization of structure-function relationships in bovine ATL1 and facilitates comparison with human disease-associated variants.