Recombinant Xenopus tropicalis ATPase family AAA domain-containing protein 1 (atad1)

What is ATAD1 and what is its functional significance in Xenopus tropicalis?

ATAD1 (ATPase family AAA domain-containing protein 1), also called THORASE, belongs to the ATPases Associated with diverse cellular Activities (AAA) superfamily of proteins that form homohexameric ring-structured complexes. It functions as a molecular machine that utilizes ATP hydrolysis to extract and remodel proteins from membranes . In Xenopus tropicalis, ATAD1 likely maintains similar core functions to its mammalian counterparts, including roles in synaptic plasticity, protein quality control, and cellular homeostasis.
The significance of ATAD1 in developmental processes makes Xenopus tropicalis an ideal model to study its function, as this organism offers advantages including external development, transparency during early stages, and a diploid genome that simplifies genetic analysis compared to the allotetraploid Xenopus laevis .

How conserved is the structure of ATAD1 across species and what structural elements define its function?

• Hexameric spiral assembly: Six ATAD1 subunits (M1-M6) rotate and translocate progressively

• Subdomain organization: Each subunit consists of a large subdomain followed by a small subdomain

• Nucleotide binding interface: ATP binds at the interface between the two subdomains

• N-terminal transmembrane region: Anchors ATAD1 to mitochondrial and peroxisomal membranes

• C-terminal AAA domain: Exposed to the cytosol, providing mechanical work via ATP hydrolysis
  Research comparing Xenopus tropicalis ATAD1 to human and yeast homologs can provide insights into evolutionary conservation of these functional domains and species-specific adaptations.

What cellular localizations and interacting partners are identified for ATAD1?

ATAD1 displays dual localization at mitochondria and peroxisomes, with the bulk of the protein typically associated with mitochondria . The subcellular distribution in Xenopus tropicalis likely mirrors this pattern, though species-specific variations may exist.
Key interacting partners identified include:

What are optimal conditions for expressing and purifying recombinant Xenopus tropicalis ATAD1?

Successful expression and purification of functional recombinant Xenopus tropicalis ATAD1 requires careful attention to maintaining the protein's ATPase activity and hexameric structure.
Recommended expression protocol:

• Expression system: E. coli BL21(DE3) or insect cell systems (for higher eukaryotic post-translational modifications)

• Expression construct: Full-length protein or truncated versions lacking the N-terminal transmembrane domain (residues ~30-413, based on human ATAD1 homology)

• Tags: N-terminal His6-tag or His6-SUMO tag with PreScission protease cleavage site

• Induction conditions: 0.1-0.5 mM IPTG at 18°C for 16-18 hours to reduce inclusion body formation

• Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM ATP, 5 mM MgCl2, 1 mM DTT, and protease inhibitors
  Purification strategy:

• IMAC chromatography (Ni-NTA)

• Tag cleavage (if using cleavable tag)

• Ion exchange chromatography

• Size exclusion chromatography in buffer containing ATP and MgCl2 to maintain hexameric assembly
  Critical considerations include maintaining ATP and magnesium in all buffers to stabilize the hexameric assembly, and using gentle detergents if purifying full-length protein with the transmembrane domain.

What assays can effectively measure ATPase activity of recombinant Xenopus tropicalis ATAD1?

Several complementary approaches can assess the ATPase activity of recombinant Xenopus tropicalis ATAD1:

• Malachite green phosphate assay:

  • Measures free inorganic phosphate released during ATP hydrolysis

  • Advantage: High sensitivity and compatibility with high-throughput screening

  • Protocol: Incubate purified ATAD1 (0.1-1 μM) with ATP (1-5 mM) in assay buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl2) at 25°C; terminate reactions at various timepoints with malachite green reagent

• Coupled enzymatic assay:

  • Links ATP hydrolysis to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Advantage: Allows real-time continuous monitoring of activity

  • Components: ATP, phosphoenolpyruvate, NADH, pyruvate kinase, and lactate dehydrogenase

• ATPase stimulation assays:

  • Measures increased activity in presence of substrates or interacting partners

  • For ATAD1, compare basal activity to activity in presence of purified substrate proteins (e.g., model tail-anchored proteins)
    These assays should be performed at physiologically relevant temperatures for Xenopus tropicalis (20-25°C) and can reveal the impact of mutations, substrate binding, or inhibitors on enzymatic activity.

How can CRISPR/Cas9 genome editing be optimized for ATAD1 studies in Xenopus tropicalis?

CRISPR/Cas9 genome editing in Xenopus tropicalis provides powerful approaches for studying ATAD1 function:

• sgRNA design considerations:

  • Target early exons to ensure complete loss-of-function

  • Select sites with minimal off-target effects using tools like CRISPRscan

  • Design multiple sgRNAs targeting different regions to increase efficiency

  • Evaluate target site conservation if aiming to replicate human disease mutations

• Delivery method:

  • Microinjection of Cas9 protein (not mRNA) with sgRNA into one-cell embryos

  • Optimal concentrations: 1-2 ng Cas9 protein with 300-500 pg sgRNA per embryo

  • Co-inject with fluorescent dextran to track injected cells

• Validation approaches:

  • T7 endonuclease assay on PCR products from F0 embryos

  • Direct sequencing of PCR products to identify indels

  • Western blotting to confirm protein loss

  • Phenotypic analysis based on expected roles of ATAD1

• Generation of stable lines:

  • Raise F0 mosaic embryos to sexual maturity (4-6 months)

  • Out-cross with wild-type animals to identify germline transmission

  • PCR genotyping of F1 offspring to identify heterozygous carriers
    This approach allows generation of both mosaic F0 animals for rapid phenotypic analysis and stable knockout lines for detailed characterization of ATAD1 function throughout development.

How does ATAD1 contribute to mitochondrial and peroxisomal protein quality control?

ATAD1 plays critical roles in quality control mechanisms for both mitochondria and peroxisomes in vertebrates, making Xenopus tropicalis an excellent model for studying these conserved functions:
In mitochondria:

• Extracts mislocalized tail-anchored (TA) proteins from the outer mitochondrial membrane

• Removes stalled import substrates, preventing mitochondrial stress

• Functions as part of a specialized membrane protein extraction system
  In peroxisomes:

• Interacts with PEX5, a cytosolic receptor for peroxisomal matrix proteins

• May participate in recycling of peroxisomal import receptors

• Could function in removal of mislocalized proteins from peroxisomal membranes
  The dual localization of ATAD1 suggests coordinated quality control mechanisms between these organelles. In Xenopus tropicalis, studying ATAD1 can reveal how these mechanisms function during embryonic development when organelle biogenesis and protein import are highly active processes.
  Research approaches should include subcellular fractionation to isolate mitochondria and peroxisomes from Xenopus tissues, combined with proteomic analysis to identify accumulated substrates in ATAD1-depleted conditions.

What is the significance of ATAD1's role in desmin intermediate filament degradation for muscle research?

ATAD1's unexpected role in muscle protein degradation offers new avenues for research using Xenopus tropicalis as a model:
ATAD1 forms a complex with PLAA and UBXN4 that catalyzes the disassembly and turnover of ubiquitinated desmin intermediate filaments (IF) . This process is critical for:

• Normal muscle plasticity and adaptation

• Prevention of abnormal protein aggregation

• Regulated muscle atrophy in response to denervation
  Unique aspects of ATAD1 function in this context include:

• Preferential activity on phosphorylated desmin substrates

• Cooperation with calpain-1 to facilitate desmin IF solubilization

• Presentation of ATAD1 as the only known AAA-ATPase with preference for phosphorylated substrates
  Xenopus tropicalis muscle development offers advantages for studying these processes, including:

• External development allowing easy observation

• Accessibility for tissue-specific manipulations

• Ability to create tissue chimeras to determine cell-autonomous versus non-autonomous effects
  A comprehensive research approach would combine ATAD1 knockout/knockdown with muscle-specific rescue, followed by analysis of muscle ultrastructure, contractile properties, and protein degradation pathways during normal development and induced atrophy.

How can research on ATAD1 mutations in Xenopus tropicalis inform therapeutic strategies for ATAD1-related human disorders?

The identification of human ATAD1 mutations causing severe neurological disorders highlights the translational potential of Xenopus tropicalis research:
Human ATAD1 mutations (e.g., p.E276X) cause a syndrome characterized by:

• Progressive extreme hypertonia

• Encephalopathy

• Seizures

• Early mortality
  Xenopus tropicalis offers unique advantages for modeling these conditions:

• Rapid development allows quick phenotypic assessment

• Optical transparency enables in vivo imaging of neural development

• Accessibility for drug treatment directly in the aqueous environment

• Ability to perform tissue-specific rescue experiments
  The therapeutic approach of AMPA receptor antagonism with perampanel proved successful in both ATAD1 knockout mice and human patients . This suggests ATAD1's critical role in AMPA receptor recycling and glutamate signaling homeostasis is evolutionarily conserved.
  Research strategy in Xenopus tropicalis:

• Generate equivalent mutations to human pathogenic variants using CRISPR/Cas9

• Characterize neural, muscle, and behavioral phenotypes

• Screen potential therapeutic compounds directly in developing embryos

• Investigate tissue-specific requirements through targeted rescue experiments
  This approach can rapidly identify evolutionarily conserved disease mechanisms and provide a platform for initial therapeutic screening before moving to mammalian models.

What approaches can resolve contradictory data regarding ATAD1 function in different cellular compartments?

Researchers investigating ATAD1 often encounter apparent contradictions regarding its localization and function across cellular compartments. These methodological approaches can help resolve such discrepancies:

• Compartment-specific tagging strategies:

  • Generate constructs with organelle-targeted ATAD1 (mitochondria-only or peroxisome-only versions)

  • Use split-protein complementation assays to detect compartment-specific interactions

  • Employ proximity labeling (BioID or APEX) to identify compartment-specific interacting partners

• Quantitative approaches:

  • Use quantitative proteomics to determine relative distribution between compartments

  • Employ superresolution microscopy with correlation analysis to measure colocalization coefficients

  • Perform immunogold electron microscopy for precise localization at ultrastructural level

• Functional separation assays:

  • Design rescue experiments with compartment-restricted ATAD1 variants

  • Measure enzymatic activity in isolated organelle fractions

  • Develop compartment-specific substrate trapping mutants

• Temporal analysis:

  • Study changes in localization during development or stress conditions

  • Use photoactivatable or photoswitchable tags to track protein movement between compartments

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure dynamics
    These approaches applied in Xenopus tropicalis can leverage the system's developmental accessibility to understand how ATAD1 function may shift between compartments during different developmental stages or physiological conditions.

What structural analysis techniques are most appropriate for studying the hexameric assembly of Xenopus tropicalis ATAD1?

Understanding the hexameric structure of ATAD1 is crucial for deciphering its function. Several complementary techniques can be employed:

• Cryo-electron microscopy (cryo-EM):

  • Particularly powerful for AAA+ ATPases like ATAD1

  • Can capture different conformational states during the ATP cycle

  • Has successfully revealed open and closed states of human ATAD1 at 3.2-3.7Å resolution

  • Required protein concentration: 1-5 mg/ml of highly pure protein

• X-ray crystallography:

  • May capture high-resolution details of specific conformational states

  • Challenging due to potential conformational heterogeneity

  • Could be successful with ATP analogs or substrate-trapped complexes

• Small-angle X-ray scattering (SAXS):

  • Provides lower resolution envelope in solution state

  • Can detect conformational changes under different nucleotide conditions

  • Complements higher resolution structural methods

• Native mass spectrometry:

  • Confirms hexameric assembly and nucleotide binding status

  • Can detect subcomplexes or alternative oligomerization states

  • Useful for studying the impact of mutations on complex stability

• Negative-stain electron microscopy:

  • Rapid assessment of sample quality and homogeneity

  • Useful preliminary step before cryo-EM

  • Can verify hexamer formation under different buffer conditions
    Combining structural data with mutagenesis of key residues identified in human ATAD1 can reveal the conservation of structural elements that specialize ATAD1 as a membrane protein extraction machine across species.

How can researchers effectively analyze the impact of ATAD1 dysfunction on neural development in Xenopus tropicalis?

Neural phenotypes associated with ATAD1 dysfunction can be comprehensively analyzed in Xenopus tropicalis using these integrated approaches:

• Morphological analysis:

  • Whole-mount in situ hybridization for neural markers

  • Immunohistochemistry for synaptic proteins

  • Time-lapse imaging of neural development in transparent embryos

  • Quantitative analysis of neural tube formation and brain development

• Functional assessment:

  • Electrophysiological recording of neural activity

  • Calcium imaging using genetically encoded indicators

  • Behavioral analysis of tadpole swimming and responses to stimuli

  • Seizure susceptibility testing using convulsant agents

• Molecular characterization:

  • RNA-seq of neural tissues from ATAD1-deficient embryos

  • Proteomics to identify accumulated ATAD1 substrates

  • Analysis of AMPA receptor levels and localization

  • Measurement of glutamate signaling components

• Mechanistic dissection:

  • Tissue-specific rescue experiments

  • Epistasis analysis with AMPA receptor modulators

  • Comparison of phenotypes with those caused by known AMPA receptor mutations

  • Temporal control of ATAD1 disruption using heat-shock or chemical-inducible systems
    These approaches can reveal whether ATAD1's role in neural development in Xenopus tropicalis aligns with its known function in AMPA receptor recycling in mammals, which when disrupted leads to hypertonia, seizures, and encephalopathy .

How do the functional characteristics of Xenopus tropicalis ATAD1 compare to its mammalian and yeast homologs?

Comparative analysis across species provides valuable insights into the evolution and conservation of ATAD1 function:

What insights can be gained from comparing ATAD1 with other AAA+ ATPases in Xenopus tropicalis?

ATAD1 belongs to the "meiotic clade" of AAA+ ATPases, and comparative analysis within Xenopus tropicalis can reveal specializations:

• Structural comparisons:

  • ATAD1 contains unique structural elements not observed in closely related family members

  • These elements likely specialize it for membrane protein extraction

  • Comparing these features across multiple Xenopus AAA+ ATPases can reveal convergent or divergent specializations

• Substrate specificity:

  • ATAD1 preferentially acts on phosphorylated substrates, a unique feature among AAA+ ATPases

  • Comparing substrate profiles across the AAA+ family in Xenopus can reveal how this specialization evolved

  • Analysis of phospho-recognition domains in various AAA+ ATPases may identify convergent mechanisms

• Developmental expression patterns:

  • Temporal expression profiling of AAA+ ATPases throughout Xenopus development

  • Spatial mapping using in situ hybridization to identify tissue-specific expression

  • Correlation of expression patterns with developmental processes requiring protein quality control

• Functional redundancy analysis:

  • Phenotypic comparison of various AAA+ ATPase knockouts

  • Double knockout combinations to identify synthetic interactions

  • Rescue experiments with chimeric proteins containing domains from different AAA+ ATPases This comparative approach can reveal how the AAA+ ATPase family has diversified in vertebrates to handle specialized functions in protein quality control across different cellular compartments and developmental contexts.