ATP5F1 Human

What is the fundamental structure and function of ATP5F1A in human mitochondria?

ATP5F1A (also known as ATP Synthase F1 Subunit Alpha) is a critical component of mitochondrial ATP synthase (Complex V), which catalyzes ATP synthesis using an electrochemical gradient of protons during oxidative phosphorylation. The ATP synthase complex consists of two linked multi-subunit complexes: the soluble catalytic core (F1) and the membrane-spanning component (Fo) comprising the proton channel .

The F1 catalytic portion contains 5 different subunits (alpha, beta, gamma, delta, and epsilon) assembled with a stoichiometry of 3 alpha, 3 beta, and a single representative of the other three subunits . ATP5F1A forms part of the catalytic core along with beta subunits, though interestingly, the alpha subunit itself does not bear the catalytic high-affinity ATP-binding sites .

During catalysis, ATP synthesis in F1 is coupled via a rotary mechanism of the central stalk subunits to proton translocation. The rotation of the central stalk against the surrounding alpha(3)beta(3) subunits leads to ATP synthesis in three separate catalytic sites on the beta subunits .

How can researchers distinguish between the various ATP5F1A isoforms in experimental settings?

ATP5F1A has multiple isoforms and aliases that researchers need to be aware of when designing experiments. The major isoforms include:

• Cardiac muscle isoform (previously known as ATP5A1) - predominantly expressed in cardiac tissue

• Non-cardiac muscle isoform (previously known as ATP5AL2) - expressed in other tissues

To distinguish between these isoforms, researchers should employ the following methodological approaches:

• RT-PCR with isoform-specific primers: Design primers that span unique exon junctions or sequence regions specific to each isoform

• Western blotting with isoform-specific antibodies: Use antibodies raised against unique epitopes in each isoform

• Mass spectrometry: Analyze tryptic peptides unique to each isoform

• RNA-seq analysis: Look for differential expression patterns across tissues

When reporting results, researchers should clearly specify which isoform nomenclature they are using, as the field has undergone several naming conventions (ATP5A, ATP5F1A, ATP5A1, etc.) . Additionally, researchers should validate antibody specificity using appropriate positive and negative controls to ensure accurate isoform identification.

What experimental approaches can assess ATP5F1 assembly into functional Complex V?

Assessing the proper assembly of ATP5F1 subunits into functional Complex V requires multiple complementary approaches:

Protein Complex Analysis:

• Blue Native PAGE (BN-PAGE): This technique separates intact protein complexes according to their molecular weight while preserving native protein-protein interactions .

• 2D BN/SDS-PAGE: Combines BN-PAGE with subsequent SDS-PAGE to identify individual subunits within complexes .

• Immunoprecipitation with subunit-specific antibodies: Allows for the isolation of intact complexes and identification of interacting partners.

Functional Assessments:

• In-gel activity assays: After BN-PAGE, gels can be incubated with ATP and lead nitrate to visualize ATP hydrolysis activity as lead phosphate precipitates.

• Spectrophotometric enzyme activity assays: Measures ATP synthesis or hydrolysis rates in isolated mitochondria.

• Oxygen consumption rate (OCR): Using platforms like Seahorse XF Analyzer to measure oligomycin-sensitive respiration, which correlates with ATP synthase function.

Structural Analysis:

• Transmission electron microscopy (TEM): Can reveal abnormalities in mitochondrial cristae structure, which often correlate with ATP synthase assembly defects .

• Immunofluorescence microscopy: Visualizes co-localization of ATP5F1 subunits within mitochondria.

In research studies, abnormal ATP synthase assembly has been observed in ATP5F1D mutations, where despite normal levels of the mutated subunit, other Complex V subunits (ATP5F1A, ATP5F1B, and ATP5PO) showed decreased abundance, indicating impaired complex assembly .

How can researchers effectively measure ATP synthase activity in relation to ATP5F1 function?

Measuring ATP synthase activity requires specialized techniques that assess both ATP production and the proton pumping function of the complex:

1. Spectrophotometric Coupled Enzyme Assays:

• ATP hydrolysis can be measured by coupling ADP production to NADH oxidation through pyruvate kinase and lactate dehydrogenase

• The decrease in NADH absorbance at 340 nm correlates with ATP hydrolysis rate

• Oligomycin sensitivity distinguishes ATP synthase activity from other ATPases

2. Luminescence-Based ATP Measurement:

• Luciferase-luciferin reactions quantify ATP production

• Can be applied to isolated mitochondria, permeabilized cells, or tissue homogenates

• Allows real-time ATP production monitoring

3. Oxygen Consumption Rate (OCR) Analysis:

• Using Seahorse XF Analyzer to measure OCR in intact cells or isolated mitochondria

• Sequential addition of oligomycin, FCCP, and rotenone/antimycin A allows calculation of:

  • ATP synthesis-linked respiration

  • Proton leak

  • Maximum respiratory capacity

  • Non-mitochondrial respiration

4. Membrane Potential Measurements:

• Fluorescent probes (TMRM, JC-1) assess the proton gradient driving ATP synthesis

• Flow cytometry or fluorescence microscopy quantifies the signal

• Provides important context when assessing ATP synthase function

When designing experiments, researchers should include appropriate controls (oligomycin inhibition), normalize to mitochondrial content (using citrate synthase activity), and account for tissue/cell-specific differences in mitochondrial content.

What are the most reliable methods for detecting ATP5F1 protein-protein interactions within the ATP synthase complex?

Detecting protein-protein interactions within the ATP synthase complex requires techniques that preserve native interactions:

1. Co-immunoprecipitation (Co-IP):

• Use antibodies against specific ATP5F1 subunits to precipitate the entire complex

• Western blot analysis of precipitated samples reveals interacting partners

• Requires careful buffer optimization to maintain interactions

• Works well for stable interactions but may miss transient ones

2. Proximity Labeling Approaches:

• BioID: Fusion of a promiscuous biotin ligase to an ATP5F1 subunit labels proximal proteins

• APEX2: Peroxidase-based proximity labeling with higher temporal resolution

• These methods capture both stable and transient interactions in living cells

• Mass spectrometry identifies labeled proteins

3. Förster Resonance Energy Transfer (FRET):

• Fluorescently tag pairs of ATP5F1 subunits (donor and acceptor)

• Energy transfer occurs only when proteins are in close proximity (1-10 nm)

• Can be measured by acceptor photobleaching or fluorescence lifetime imaging

• Allows for real-time monitoring of interactions in living cells

4. Crosslinking Mass Spectrometry (XL-MS):

• Chemical crosslinkers create covalent bonds between interacting proteins

• MS/MS analysis identifies crosslinked peptides

• Provides detailed spatial information about interaction interfaces

• Particularly valuable for mapping the rotary mechanism of ATP synthase

5. Cryo-Electron Microscopy:

• Visualizes the entire ATP synthase complex at near-atomic resolution

• Can reveal structural details of subunit interactions

• When combined with site-directed mutagenesis, can validate interaction models

Research has shown that pathogenic variants in ATP5F1D (p.Pro82Leu and p.Val106Gly) do not affect protein levels but disrupt binding to other F1 subunits, demonstrating how these techniques can reveal mechanistic insights into disease-causing mutations .

What strategies can overcome challenges in isolating and purifying functional ATP5F1 subunits?

Isolating functional ATP5F1 subunits presents several technical challenges that researchers must address:

1. Optimized Solubilization Strategies:

• Gentle detergents: Digitonin (0.5-1%) or n-dodecyl-β-D-maltoside (0.5-1%) preserve native structures

• Gradual solubilization at low temperatures (4°C) minimizes denaturation

• Buffer optimization (pH 7.2-7.8, 150-250 mM NaCl) maintains stability

• Addition of ATP or ADP (1-2 mM) can stabilize conformations

2. Advanced Purification Approaches:

• Affinity chromatography using:

  • Histidine-tagged recombinant subunits

  • ATP-agarose for nucleotide-binding subunits

  • Specific antibody columns

• Blue Native electrophoresis followed by electroelution for intact complexes

• Size exclusion chromatography to separate fully assembled complexes from subcomplexes

3. Expression Systems Considerations:

• Mammalian expression systems (HEK293T, CHO) maintain proper folding and modifications

• Co-expression of multiple subunits improves stability and assembly

• Inducible expression systems with temperature control prevent toxicity

• Addition of specific chaperones can improve yield of properly folded proteins

4. Stability Enhancement Approaches:

• Addition of stabilizing agents:

  • 10-15% glycerol

  • 1-5 mM DTT or TCEP to maintain reduced cysteines

  • Protease inhibitor cocktails

• Chemical crosslinking for structural studies

• Nanodiscs or amphipols to maintain membrane protein stability

5. Functional Validation Methods:

• ATPase activity assays to confirm enzymatic function

• Reconstitution into liposomes to assess proton pumping

• Structural analysis by circular dichroism and thermal shift assays

When purifying ATP5F1D specifically, researchers have observed that disease-associated variants (p.Pro82Leu and p.Val106Gly) affect interaction with other F1 subunits but not protein stability, highlighting the importance of assessing both protein levels and functional interactions .

How do mutations in ATP5F1 genes contribute to mitochondrial disease phenotypes?

Mutations in ATP5F1 genes can lead to severe mitochondrial diseases through several pathogenic mechanisms:

1. Impaired ATP Synthase Assembly:
Biallelic missense variants in ATP5F1D (p.Pro82Leu and p.Val106Gly) have been shown to cause a Mendelian mitochondrial disease characterized by episodic metabolic decompensation, lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia . Functional studies demonstrated that these mutations do not affect ATP5F1D protein levels but disrupt its ability to interact with other F1 subunits, leading to reduced complex V assembly .

2. Structural Mitochondrial Abnormalities:
Transmission electron microscopy of fibroblasts from patients with ATP5F1D mutations revealed dramatically decreased cristae number . In induced pluripotent stem cell (iPSC)-derived cardiomyocytes, both mitochondrial size and cristae number were reduced . These structural abnormalities directly impact the capacity for oxidative phosphorylation.

3. Bioenergetic Dysfunction:
Patient-derived cells carrying ATP5F1D mutations exhibited impaired maximal respiration, particularly in response to metabolic substrates like palmitate . This functional respiratory deficit translates to insufficient ATP production, especially during periods of increased energy demand.

4. Tissue-Specific Manifestations:
The clinical presentation of ATP5F1 mutations often shows tissue specificity, with energy-demanding tissues like the brain, heart, and skeletal muscle being most affected. This reflects the different energy requirements and mitochondrial content across tissues.

A particularly effective experimental approach for validating pathogenicity is the use of model organisms. Knockdown of the ATP5F1D homolog (ATPsynδ) in Drosophila caused a near-complete loss of the fly head, and this severe phenotype could be rescued by wild-type human ATP5F1D but not by the mutant versions, confirming their pathogenicity .

What is the relationship between ATP5F1 expression and cancer development?

The relationship between ATP5F1 expression and cancer involves complex metabolic reprogramming:

1. Altered Expression Patterns:
Studies have systematically analyzed the expression of mitochondrial ATP synthase (Complex V) subunits in clear cell renal cell carcinoma (ccRCC) . ATP5F1A and other complex V subunits show altered expression in various cancers, potentially serving as biomarkers for diagnosis or prognosis .

2. Metabolic Reprogramming Mechanisms:
Cancer cells often exhibit the Warburg effect - a shift from oxidative phosphorylation to aerobic glycolysis despite oxygen availability. Changes in ATP synthase subunit expression may either:

• Drive this metabolic shift by limiting oxidative phosphorylation

• Occur as a consequence of metabolic adaptation to the tumor microenvironment

• Represent a compensatory response to altered energy demands

3. Beyond Energy Production:
ATP synthase components have roles beyond ATP production that may influence cancer biology:

• Involvement in apoptosis regulation (particularly through interactions with mitochondrial permeability transition pore)

• Potential roles in cell signaling pathways

• Contributions to mitochondrial morphology and dynamics

4. Research Approaches:
To study ATP5F1 in cancer contexts, researchers employ:

• Expression analysis comparing tumor vs. adjacent normal tissue

• Correlation of expression levels with clinical parameters and outcomes

• Knockdown/overexpression studies in cancer cell lines

• Metabolic flux analysis to assess bioenergetic consequences

5. Therapeutic Implications:
Understanding ATP5F1 dysregulation in cancer could lead to:

• Novel diagnostic or prognostic biomarkers

• Potential therapeutic targets

• Strategies to target cancer-specific metabolic vulnerabilities

The systematic analysis of ATP synthase subunit expression in ccRCC represents an important step toward developing better understanding of this complex's role in cancer biology .

How do post-translational modifications of ATP5F1 subunits impact mitochondrial function?

Post-translational modifications (PTMs) of ATP5F1 subunits represent a critical regulatory mechanism:

1. Types of PTMs Affecting ATP5F1 Subunits:

2. Functional Consequences of PTMs:

• Altered catalytic efficiency of ATP synthesis

• Modified assembly/disassembly kinetics of the complex

• Changed stability of individual subunits

• Altered interactions between subunits

• Modified responses to regulatory signals

3. Methods for Studying ATP5F1 PTMs:

Mass Spectrometry Approaches:

• Enrichment strategies for specific PTMs (phosphopeptides, acetylated peptides)

• Parallel reaction monitoring for targeted analysis

• SILAC or TMT labeling for quantitative comparisons

• PTM crosstalk analysis using multi-protease digestion

Functional Validation:

• Site-directed mutagenesis of modified residues

• Expression of phosphomimetic/phospho-dead mutants

• In vitro treatment with modifying/demodifying enzymes

4. Disease Implications:
PTM dysregulation can contribute to pathology through:

• Hyperphosphorylation or hyperacetylation altering ATP synthase function

• Oxidative stress leading to irreversible modifications

• Disrupted PTM regulatory networks affecting mitochondrial adaptation

Studying these modifications provides insights into how ATP synthase function is fine-tuned in health and dysregulated in disease, potentially revealing novel therapeutic targets for mitochondrial dysfunction.

How can CRISPR-Cas9 gene editing be optimized for studying ATP5F1 subunit functions?

CRISPR-Cas9 technology offers powerful approaches for studying ATP5F1 subunits, but requires careful optimization:

1. Strategic Design Considerations:

Guide RNA Design:

• Target specificity is critical due to homology between ATP5F1 subunits

• Off-target analysis using tools like CRISPOR or Cas-OFFinder

• Design multiple gRNAs per target and validate efficiency

• Consider chromatin accessibility at target sites

Editing Strategies:

• Complete knockout may be lethal - consider conditional approaches

• Knockin of specific mutations (e.g., ATP5F1D p.Pro82Leu) to model patient variants

• Epitope tagging for visualization and purification

• Fluorescent protein fusions for live imaging

2. Delivery Optimization:

3. Advanced CRISPR Applications:

• Base editing: For precise nucleotide changes without double-strand breaks

• Prime editing: For precise insertions, deletions, and all base-to-base conversions

• CRISPRi/CRISPRa: For modulation of gene expression without DNA modification

• CRISPR screening: For identifying genetic interactions with ATP5F1 subunits

4. Validation Approaches:

• Western blotting to confirm protein knockout/modification

• BN-PAGE to assess complex assembly

• Functional assays (OCR, ATP production)

• Electron microscopy for cristae morphology analysis

• Rescue experiments with wild-type cDNA to confirm specificity

5. Cell Model Selection:

• Patient-derived fibroblasts for studying natural mutations

• iPSCs for differentiation into affected cell types (e.g., cardiomyocytes, neurons)

• Cell types with high mitochondrial content show more pronounced phenotypes

CRISPR-based studies have been instrumental in confirming pathogenicity of ATP5F1D variants, as demonstrated by rescue experiments in model organisms .

What computational approaches best predict the impact of novel ATP5F1 variants?

Computational prediction of ATP5F1 variant effects requires multiple complementary approaches:

1. Sequence-Based Methods:

• Conservation Analysis: Highly conserved residues are more likely functionally important

• Variant Effect Predictors:

  • SIFT: Predicts effects based on sequence homology

  • PolyPhen-2: Combines sequence conservation with structural features

  • CADD: Integrates multiple annotations into a single pathogenicity score

  • Ensemble methods (REVEL, VEST) often outperform individual predictors

2. Structure-Based Methods:

• Protein Stability Analysis:

  • FoldX: Calculates changes in folding free energy (ΔΔG)

  • Rosetta: Models structural perturbations

  • DUET: Integrates multiple stability predictors

• Molecular Dynamics Simulations:

  • Simulates atomic movements over time

  • Reveals potential disruptions to subunit interactions and conformational changes

  • Particularly valuable for the rotary mechanism of ATP synthase

3. ATP Synthase-Specific Approaches:

• Interface Analysis: Examining subunit interaction interfaces is critical, as demonstrated by ATP5F1D mutations that disrupt binding to other F1 subunits

• Functional Domain Mapping: Identifying variants in regions critical for:

  • Nucleotide binding

  • Catalytic activity

  • Rotary mechanism

  • Subunit assembly

4. Validation Against Known Disease Mutations:

The p.Pro82Leu and p.Val106Gly variants in ATP5F1D provide valuable benchmarks for computational prediction methods . These variants don't affect protein stability but disrupt complex assembly, highlighting the importance of considering protein-protein interactions in predictions.

When applying computational approaches to novel ATP5F1 variants, researchers should:

• Use multiple complementary methods rather than relying on a single predictor

• Consider the specific structural and functional context of the variant

• Validate predictions with experimental approaches when possible

• Be cautious about variants in regions with limited structural information

How can multi-omics approaches integrate data to understand ATP5F1 dysfunction?

Multi-omics integration provides comprehensive insights into ATP5F1 dysfunction:

1. Multi-Omics Data Collection:

2. Integration Strategies:

Network-Based Approaches:

• Construct multi-layered networks connecting genetic variants to functional outcomes

• Identify key nodes that connect different data types

• Tools: Cytoscape with OmicsIntegrator, NetworkAnalyst

Machine Learning Integration:

• Dimensionality reduction using MOFA (Multi-Omics Factor Analysis)

• Patient stratification based on integrated profiles

• Predictive modeling of disease progression

Pathway-Centered Analysis:

• Map all omics data onto oxidative phosphorylation pathways

• Identify regulatory relationships between omics layers

• Use ATP5F1 as focal point for integrative analysis

Case Study: ATP5F1D Mutations

In the case of ATP5F1D mutations causing mitochondrial disease , a multi-omics approach revealed:

• Genomics: Identified causal variants (p.Pro82Leu and p.Val106Gly)

• Proteomics: Showed normal ATP5F1D levels but decreased other complex V subunits

• Structural biology: Revealed decreased cristae

• Functional assays: Demonstrated impaired respiration

• Model organisms: Confirmed pathogenicity through rescue experiments

This comprehensive approach provided mechanistic understanding from gene to phenotype, demonstrating how multiple data types collectively explain the disease process.

4. Implementation Considerations:

• Begin with hypothesis-driven core questions about ATP5F1 function

• Carefully plan experimental design to enable integration

• Consider time-course data to capture dynamic processes

• Include appropriate controls at each omics layer

• Validate key findings using orthogonal methods

Multi-omics approaches are particularly valuable for understanding complex mitochondrial diseases where ATP5F1 dysfunction may be just one component of broader bioenergetic dysregulation.