ATP synthase 6 kDa subunit, mitochondrial Antibody

What is the fundamental role of ATP synthase in mitochondrial function?

ATP synthase (Complex V) plays a critical role in energy metabolism, serving as the final component of the oxidative phosphorylation system. This transmembrane enzyme is responsible for converting adenosine diphosphate (ADP) to adenosine triphosphate (ATP), the primary energy currency of cells. The enzyme contains multiple subunits that work together to harness the proton gradient across the inner mitochondrial membrane, driving the synthesis of ATP through a rotary mechanism . Mitochondria produce energy through oxidative phosphorylation, using oxygen and simple sugars to create ATP that can be utilized for cellular processes .

How is the ATP synthase complex organized structurally?

ATP synthase consists of two main domains: the F₁ catalytic domain that protrudes into the mitochondrial matrix and the F₀ membrane domain embedded in the inner mitochondrial membrane. The complex contains multiple subunits, including:

• F₁ domain: Alpha (55 kDa), Beta (ATP5B, ~55-60 kDa), and other regulatory subunits

• F₀ domain: Includes subunit c (ATP5G) , subunit d (ATPd) , and ATP6 (encoded by MT-ATP6 gene)

These subunits assemble into a functional holoenzyme that couples proton flow with ATP synthesis. The beta subunit contains the catalytic site responsible for ATP production, while other subunits play structural and regulatory roles in the complex .

What is the relationship between MT-ATP6 gene and ATP synthase functionality?

The MT-ATP6 gene encoded in mitochondrial DNA provides instructions for making a protein essential for normal ATP synthase function. This protein forms a critical subunit of the F₀ portion of ATP synthase. Mutations in the MT-ATP6 gene can impair the function or stability of the ATP synthase complex, inhibiting ATP production and disrupting oxidative phosphorylation . Such impairments have been linked to mitochondrial diseases, including Leigh syndrome, which affects approximately 10% of patients with mutations in this gene .

What criteria should guide selection of antibodies for ATP synthase subunit research?

When selecting antibodies for ATP synthase research, researchers should consider:

• Specificity: Confirm the antibody targets the specific subunit of interest without cross-reactivity

• Host species: Select based on experimental design and compatibility with secondary detection systems

• Application compatibility: Ensure the antibody is validated for your specific application (Western blot, immunohistochemistry, etc.)

• Clone information: For monoclonal antibodies, know the specific clone (e.g., 6G11 for ATP5B)

• Immunogen details: For polyclonal antibodies, understand the specific peptide sequence used as immunogen

For example, when studying the beta subunit, researchers might select a polyclonal antibody like AS16 3976, which is raised against KLH-conjugated synthetic peptides derived from plant and algal mitochondrial sequences of beta subunits of F-type ATP synthases .

How can researchers validate the specificity of ATP synthase subunit antibodies?

Validation of antibody specificity requires multiple complementary approaches:

• Western blot analysis: Confirm single band detection at the expected molecular weight (e.g., ~55 kDa for beta subunit, 59.6 kDa theoretical/55 kDa apparent)

• Knockout/knockdown controls: Use RNAi or CRISPR to reduce expression and verify corresponding reduction in antibody signal

• Multiple antibody comparison: Use antibodies against different epitopes of the same protein to confirm consistent detection patterns

• Recombinant protein controls: Test against purified recombinant proteins when available

• Mass spectrometry validation: Confirm identity of detected proteins by mass spectrometry following immunoprecipitation

For example, RNAi knockdown of ATP synthase subunit d (ATPd) reduced protein levels to 10-25% of wild-type levels, confirming antibody specificity in these experimental systems .

What are key differences between monoclonal and polyclonal antibodies for ATP synthase research?

What are optimal protocols for mitochondrial protein extraction to study ATP synthase?

Effective mitochondrial protein extraction is critical for ATP synthase research:

• Tissue preparation: Fresh or flash-frozen tissue should be homogenized in isolation buffer (typically containing sucrose, HEPES, EDTA, and protease inhibitors)

• Differential centrifugation: Sequential centrifugation steps (600-1000g to remove nuclei, 10,000-12,000g to pellet mitochondria)

• Purification: Further purification can be achieved using density gradient centrifugation

• Protein extraction: Mitochondrial proteins can be solubilized using:

  • 2% SDS for denaturing conditions

  • Digitonin (0.5-2%) or n-dodecyl β-D-maltoside (0.5-1%) for native conditions

• Protein quantification: Bradford or BCA assay to standardize loading

For example, in studies with cauliflower (Brassica oleracea var. botrytis), researchers isolated mitochondrial proteins and denatured them with a standard sample buffer containing 2% SDS, 10% glycerol, 50 mM Tris-HCl pH 6.8, 0.1% bromophenol blue, and 1% β-mercaptoethanol at 80°C for 10 minutes prior to Western blot analysis .

How should researchers optimize Blue Native-PAGE for ATP synthase complex analysis?

Blue Native-PAGE (BN-PAGE) is essential for studying intact ATP synthase complexes:

• Sample preparation:

  • Solubilize mitochondrial membranes with gentle detergents (digitonin for supercomplexes, n-dodecyl β-D-maltoside for individual complexes)

  • Maintain 4°C throughout preparation

  • Add Coomassie Blue G-250 to provide negative charge

• Gel preparation:

  • Use gradient gels (typically 3-12% or 4-16% acrylamide)

  • Add Coomassie Blue to cathode buffer initially

• Electrophoresis conditions:

  • Run at low voltage (50-100V) initially, then increase to 200-300V

  • Consider switching to cathode buffer without Coomassie partway through

• Detection methods:

  • Western blot using antibodies against specific subunits

  • In-gel activity assays for ATP synthase

  • Second dimension SDS-PAGE for subunit composition analysis

This technique has been successfully applied to evaluate how decreased ATP synthase subunit d affects assembly of the ATP synthase complex and other OXPHOS complexes .

What methodological approaches are effective for measuring ATP synthase activity?

Several complementary methods can be used to assess ATP synthase activity:

• ATP production assays:

  • Luciferase-based assays measuring ATP production in isolated mitochondria

  • Oxygen consumption measurements using Clark-type electrodes or Seahorse analyzers

  • High ATP-producing cells show increased proliferation, stemness, and migration capabilities

• In-gel activity assays:

  • Following BN-PAGE, gels can be incubated in buffer containing ATP, lead nitrate, and magnesium

  • ATP hydrolysis by ATP synthase results in lead phosphate precipitation visible as white bands

• ATP synthase inhibition studies:

  • Oligomycin as a classical inhibitor of ATP synthase

  • Bedaquiline as a newer ATP synthase inhibitor that downregulates ATP5F1C expression and prevents metastasis in cancer models

• Membrane potential measurements:

  • Fluorescent dyes like TMRM or JC-1 to assess mitochondrial membrane potential

  • Correlating membrane potential with ATP synthase activity

Research demonstrates that ATP-high cancer cells show a nearly fivefold increase in metastatic capacity in vivo, highlighting the importance of accurately measuring ATP synthase activity in different experimental contexts .

How does ATP synthase dysfunction contribute to disease pathology?

ATP synthase dysfunction has been implicated in several pathological conditions:

• Mitochondrial diseases:

  • Mutations in MT-ATP6 cause approximately 10% of Leigh syndrome cases

  • T8993G mutation in MT-ATP6 impairs ATP synthase function, leading to decreased ATP production

  • Symptoms include developmental delay, muscle weakness, movement problems, and breathing difficulties

• Cancer progression:

  • High ATP production by mitochondrial ATP-synthase promotes cancer progression

  • ATP-high cancer cells show increased proliferation, stemness, anchorage-independence, migration, invasion, and multi-drug resistance

  • ATP5F1C (gamma-subunit) overexpression correlates with metastatic potential

• Cellular energy deficiency:

  • Impaired oxidative phosphorylation from ATP synthase dysfunction can lead to cell death due to energy depletion

  • Tissues with high energy demands (brain, muscles, heart) are particularly vulnerable

• Mitochondrial stress response:

  • ATP synthase inhibition induces mitochondrial dysfunction signaling genes

  • This triggers adaptive cellular responses that can influence disease progression

How can ATP synthase be targeted therapeutically in disease contexts?

Recent research has identified several strategies for targeting ATP synthase therapeutically:

• Cancer treatment approaches:

  • Bedaquiline (FDA-approved drug) downregulates ATP5F1C expression in vitro and prevents spontaneous metastasis in vivo

  • Targeting ATP-high cancer cell populations may prevent tumor progression and metastasis

  • Importantly, Bedaquiline showed no effect on non-tumorigenic mammary epithelial cells or primary tumors, suggesting specificity for metastatic processes

• Mitochondrial disease interventions:

  • Gene therapy approaches targeting mutant MT-ATP6

  • Small molecules that enhance ATP synthase assembly or stability

  • Metabolic bypass strategies to compensate for ATP deficiency

• Biomarker development:

  • ATP5F1C is a promising biomarker for metastasis prediction

  • Immunohistochemical detection of ATP5F1C protein expression correlates with metastatic potential

  • Metastasis gene signature includes mitochondrial-related genes, particularly ATP5F1C

Research indicates that mitochondrial ATP depletion represents a promising therapeutic strategy for metastasis prophylaxis and addressing treatment failure in cancer .

What methodological considerations are important when studying ATP synthase in cancer models?

When investigating ATP synthase in cancer research, several methodological considerations are critical:

• Cell population heterogeneity:

  • Cancer cell populations contain ATP-high and ATP-low subpopulations with different properties

  • Methodologies must account for this heterogeneity (cell sorting, single-cell analyses)

  • ATP-high cells demonstrate specific phenotypic characteristics (increased proliferation, stemness, migration)

• In vivo validation requirements:

  • Cell culture findings must be validated in animal models

  • ATP-high MDA-MB-231 cells showed approximately fivefold increase in metastatic capacity in vivo

  • Therapeutic administration of ATP synthase inhibitors should be tested for effects on both primary tumors and metastasis

• Molecular marker assessment:

  • ATP-high cells overexpress components of mitochondrial complexes I-V

  • These cells also express markers associated with circulating tumor cells and metastasis (EpCAM, VCAM1)

  • Comprehensive profiling should include both mitochondrial and metastasis-related markers

• Functional assays:

  • Anchorage-independent growth assays

  • Cell migration and invasion assays

  • Multi-drug resistance testing

  • Antioxidant capacity measurements

Knockdown of ATP5F1C significantly reduces ATP production, anchorage-independent growth, and cell migration, highlighting the importance of targeting specific ATP synthase subunits in experimental approaches .

How can researchers address antibody cross-reactivity issues in ATP synthase studies?

Cross-reactivity can complicate ATP synthase research. Consider these approaches:

• Pre-absorption controls:

  • Incubate antibody with immunizing peptide before application

  • Should eliminate specific signal while leaving non-specific binding

• Multiple antibody validation:

  • Use antibodies from different sources targeting different epitopes

  • Compare reactivity patterns across experimental conditions

• Species-specific considerations:

  • Be aware of species differences in ATP synthase structure

  • Some antibodies show predicted reactivity across species (e.g., AS16 3976 expected to work with Chlamydomonas reinhardtii, Nicotiana tabacum, Oryza sativa, Phaeodactylum tricornutum)

  • Others fail to detect homologs in certain species (e.g., not reactive in Dunaliella salina)

• Dilution optimization:

  • Titrate antibody concentrations (e.g., 1:1000-1:5000 for Western blot applications)

  • Higher dilutions often reduce background/non-specific binding

• Blocking optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Match blocking agent to application and detection system

What approaches can resolve difficulties in detecting low-abundance ATP synthase subunits?

Detection of low-abundance ATP synthase subunits requires specialized techniques:

• Sample enrichment:

  • Isolate pure mitochondrial fractions to concentrate mitochondrial proteins

  • Consider immunoprecipitation to enrich specific subunits

• Detection system optimization:

  • Use high-sensitivity chemiluminescent substrates for Western blot

  • Consider fluorescent secondary antibodies for improved quantification

  • Amplification systems (biotin-streptavidin, tyramide signal amplification)

• Loading optimization:

  • Increase protein loading (e.g., from 10 µg to 20 µg of mitochondrial proteins)

  • Balance increased loading with potential resolution problems

• Exposure time adjustment:

  • Optimize exposure times to detect faint bands

  • Use internal controls to normalize across experiments

• Alternative detection methods:

  • Mass spectrometry-based approaches for very low abundance subunits

  • RT-qPCR to assess transcript levels as a proxy for protein expression

How can researchers effectively study ATP synthase in the context of mitochondrial stress responses?

Studying ATP synthase during mitochondrial stress requires integrated approaches:

• Gene expression analysis:

  • Use qRT-PCR to monitor mitochondrial dysfunction signaling (MDS) genes

  • Include genes like AOX1s, UPOX1, MGE1, NDB4, OM66, HSP23.5, ANAC013, and SOT12

  • Compare expression patterns between wild-type and ATP synthase-deficient models

• ROS measurement integration:

  • Combine ATP synthase studies with ROS detection methods

  • ANAC013 transcription factor translocates to nucleus upon sensing mitochondrial ROS

  • Correlate ROS levels with ATP synthase dysfunction

• Mitochondrial morphology assessment:

  • ATP synthase dysfunction may alter mitochondrial ultrastructure

  • Combine biochemical approaches with electron microscopy

• Genetic manipulation approaches:

  • RNAi knockdown of specific subunits (e.g., ATPd knockdown to 10-25% of wild-type levels)

  • Analyze effects on complex assembly, ATP production, and cell physiology

  • Verify that knockdown of one subunit doesn't affect expression of other subunits at transcriptional level

• Blue Native-PAGE analysis:

  • Assess how decreased expression of specific subunits affects assembly of ATP synthase complex

  • Determine whether other OXPHOS complexes are affected by ATP synthase dysfunction

By integrating these approaches, researchers can develop a comprehensive understanding of how ATP synthase participates in and responds to mitochondrial stress conditions.