Recombinant Vanderwaltozyma polyspora ATP synthase subunit 9, mitochondrial (ATP9)

What is Vanderwaltozyma polyspora ATP synthase subunit 9, mitochondrial (ATP9)?

ATP synthase subunit 9 is a critical component of the mitochondrial ATP synthase complex in Vanderwaltozyma polyspora (also known as Kluyveromyces polysporus). The protein is encoded by the ATP9 gene (ORF name: VapofMp02) and has a UniProt accession number of A6H4Q2. It functions as part of the F0 portion of ATP synthase, which is responsible for proton translocation across the inner mitochondrial membrane. This protein also has an alternative designation as a lipid-binding protein, suggesting additional functional roles beyond ATP synthesis .

What is the amino acid composition and sequence of V. polyspora ATP9?

The V. polyspora ATP9 protein consists of 76 amino acids with the following sequence: MQLVLAAKYIGAGISTIGLLGAGIGIAIVFAALINGVSRNPSLRETLFPMAILGFALSEAATGLFCLMISFLLIYAV. The protein exhibits characteristic hydrophobic transmembrane regions typical of proteins embedded in the mitochondrial inner membrane. The expression region spans positions 1-76, representing the full-length protein .

What are the optimal storage conditions for recombinant ATP9 protein?

For optimal stability and activity maintenance of recombinant V. polyspora ATP9:

• Store at -20°C for routine use

• For extended preservation, maintain at either -20°C or -80°C

• Working aliquots can be kept at 4°C for up to one week

• The protein is typically supplied in a Tris-based buffer containing 50% glycerol optimized for this specific protein

• Avoid repeated freeze-thaw cycles, as they can compromise protein integrity and functionality

How does V. polyspora ATP9 compare to homologous proteins in other yeast species?

V. polyspora shares evolutionary relationships with other yeast species such as Candida glabrata and Ashbya gossypii, suggesting conservation of mitochondrial functionality across these organisms . Comparative analysis of ATP9 across yeast species reveals structural and functional conservation, particularly in membrane-spanning regions essential for proton translocation. When designing experiments involving V. polyspora ATP9, researchers should consider these evolutionary relationships, especially when extrapolating findings from better-characterized yeast models like Saccharomyces cerevisiae.

How can recombinant ATP9 be used to study mitochondrial dysfunction mechanisms?

Recombinant V. polyspora ATP9 serves as a valuable tool for investigating mitochondrial dysfunction through several methodological approaches:

• Comparative proteomics: Use recombinant ATP9 as a standard to quantify expression levels in experimental samples

• Functional reconstitution: Incorporate the protein into liposomes to study proton translocation capacity

• Interaction studies: Employ the recombinant protein to identify binding partners altered during mitochondrial stress

• Structural analysis: Use purified ATP9 for crystallography or cryo-EM studies to examine conformational changes

Research has demonstrated connections between mitochondrial dysfunction and the retrograde response pathway, where communication between mitochondria and the nucleus regulates cellular adaptation to respiratory deficiencies . Changes in ATP synthase components like ATP9 can trigger this response, making recombinant ATP9 useful for mechanistic studies of this pathway.

What role might ATP9 play in mitochondrial genome stability and inheritance?

Studies have revealed connections between mitochondrial function and genome stability that may involve ATP9:

• Loss of respiratory function correlates with the formation of Extrachromosomal rDNA Circles (ERCs) in yeast

• Respiratory incompetence results in altered silencing patterns at the rDNA locus

• ATP synthase components may influence mitochondrial DNA inheritance patterns

Methodological approach for investigating ATP9's role in mitochondrial inheritance:

• Use recombinant ATP9 in binding assays with mitochondrial nucleoids

• Compare petite frequency in wild-type versus ATP9-mutant strains

• Analyze mitochondrial segregation patterns during cell division

• Examine the influence of ATP9 variants on mitochondrial genome transmission during mating

How can researchers examine ATP9's potential involvement in the retrograde response pathway?

The retrograde response is a signaling pathway activated upon mitochondrial dysfunction, with potential connections to ATP9 function:

Experimental methodology:

• Generate ATP9 knockdown or mutant strains

• Monitor retrograde response gene expression using RT-qPCR

• Analyze changes in rDNA organization and ERC formation following ATP9 manipulation

• Perform epistasis analysis with known retrograde response components

• Assess changes in mitochondrial membrane potential and respiratory capacity

Research has shown that respiratory incompetence can lead to the formation of self-replicating Extrachromosomal rDNA Circles (ERCs) and altered gene expression patterns , suggesting ATP9 dysfunction might trigger retrograde signaling cascades.

What experimental challenges are commonly encountered when working with ATP9 and how can they be addressed?

How does ATP9 participate in mitochondrial bioenergetics and what methods can assess its function?

ATP9 forms the c-ring of the F0 portion of ATP synthase, creating a channel for proton translocation that drives ATP synthesis. To assess its functional activity:

• Proton translocation assays: Measure the ability of reconstituted ATP9 to facilitate proton movement across membranes using pH-sensitive dyes

• ATP synthesis measurements: Quantify ATP production in reconstituted systems containing purified ATP9

• Membrane potential analysis: Evaluate the contribution of ATP9 to establishing and maintaining mitochondrial membrane potential

• Oligomerization studies: Assess the ability of ATP9 to form proper c-ring structures using crosslinking and native gel electrophoresis

• Inhibitor sensitivity profiling: Compare the effects of known ATP synthase inhibitors on systems containing wild-type versus mutant ATP9

Research on mitochondrial petite mutants has demonstrated that impaired respiratory function affects both catabolic and anabolic metabolism , highlighting the critical role of ATP synthase components like ATP9 in cellular bioenergetics.

What approaches can be used to study potential interactions between ATP9 and other mitochondrial proteins?

To investigate protein-protein interactions involving ATP9:

• Co-immunoprecipitation: Using antibodies against ATP9 or potential interacting partners

• Proximity labeling: Employing BioID or APEX2 fusions to identify proteins in close proximity to ATP9

• Crosslinking mass spectrometry: Capturing transient interactions through chemical crosslinking

• Two-hybrid screening: Yeast or bacterial two-hybrid systems adapted for membrane proteins

• FRET/BRET analysis: Measuring fluorescence or bioluminescence resonance energy transfer between tagged proteins

Previous research has identified interactions between mitochondrial proteins and components involved in coenzyme Q biosynthesis , suggesting similar approaches could reveal ATP9 interaction partners.

How can researchers validate the functional integrity of recombinant ATP9?

Ensuring recombinant ATP9 maintains its native structure and function is critical for reliable experimental results:

Validation workflow:

• Structural analysis: Circular dichroism to confirm secondary structure elements

• Thermal stability: Differential scanning fluorimetry to assess protein stability

• Membrane integration: Flotation assays to verify proper membrane incorporation

• Oligomeric state: Blue native PAGE to examine complex formation

• Functional reconstitution: Proteoliposome-based assays measuring proton translocation

For functional complementation studies, researchers can test the ability of recombinant ATP9 to rescue phenotypes in ATP9-deficient yeast strains, measuring growth rates under respiratory conditions and respiratory complex assembly.

What controls should be included in experiments involving recombinant V. polyspora ATP9?

For robust experimental design:

• Positive controls: Well-characterized ATP synthase subunits from model organisms like S. cerevisiae

• Negative controls: Inactive ATP9 mutants with altered critical residues

• Specificity controls: Related but functionally distinct membrane proteins

• Expression controls: Normalization to housekeeping genes when measuring expression

• Technical controls: Accounting for detergent effects in membrane protein assays

Quantitative controls for recombinant protein verification:

• Concentration standards for western blotting

• Purified ATP synthase complexes for functional benchmarking

• Mock-transfected/transformed controls for expression systems

How can V. polyspora ATP9 be used to study mitochondrial genome evolution?

The evolutionary relationship between V. polyspora and other yeast species like Candida glabrata and Ashbya gossypii makes ATP9 valuable for studying mitochondrial evolution:

• Comparative genomics: Analyze sequence conservation and selection pressure on ATP9 across yeast lineages

• Functional complementation: Test cross-species functionality by expressing V. polyspora ATP9 in other yeast species

• Co-evolution analysis: Examine coordinated evolution between ATP9 and other ATP synthase components

• Horizontal gene transfer: Investigate potential genetic exchange events involving ATP9

Research has shown that mitochondrial functionality affects nuclear genome stability through mechanisms like ERC formation , highlighting the complex evolutionary interplay between mitochondrial proteins like ATP9 and nuclear genome maintenance.

What techniques can assess the impact of ATP9 variants on mitochondrial function?

To evaluate how variations in ATP9 sequence affect mitochondrial function:

• Site-directed mutagenesis: Generate specific ATP9 variants based on evolutionary or clinical interest

• Heterologous expression: Express variants in model systems like S. cerevisiae

• Respiratory capacity measurement: Assess oxygen consumption rates in cells expressing different variants

• Membrane potential analysis: Measure mitochondrial membrane potential using fluorescent dyes

• Petite frequency determination: Quantify the rate of respiratory-deficient colony formation

Experiments investigating mitochondrial function have demonstrated that respiratory incompetence affects both mitochondrial DNA stability and nuclear gene expression , providing a framework for studying ATP9 variant effects.

How should researchers interpret contradictory results in ATP9 functional studies?

When facing inconsistent results:

• Consider context dependency: ATP9 function may vary with lipid environment, pH, or ionic conditions

• Examine protein quality: Verify proper folding and oligomerization state of the recombinant protein

• Assess experimental system limitations: Different reconstitution methods may yield varying results

• Compare across species: Results from V. polyspora ATP9 may differ from homologs in other species

• Evaluate technical variables: Detergent choice, buffer composition, and temperature can significantly impact results

Methodology for resolving contradictions:

• Perform side-by-side comparisons under identical conditions

• Use multiple complementary techniques to assess the same parameter

• Validate findings in different experimental systems (in vitro, ex vivo, in vivo)

What are common pitfalls in experimental design involving recombinant ATP9?

Researchers should be aware of these potential issues:

• Detergent interference: Detergents used for solubilization may affect activity measurements

• Incomplete complex formation: ATP9 functions optimally within the complete ATP synthase complex

• Post-translational modification differences: Recombinant systems may not reproduce native modifications

• Buffer compatibility: Ion concentrations critical for ATP9 function must be carefully controlled

• Storage degradation: Improper storage can lead to protein aggregation or denaturation

Methodological solutions include optimizing purification protocols, carefully selecting appropriate detergents, and implementing rigorous quality control testing before functional experiments.