ATP5I Antibody

What is ATP5I and what is its role in mitochondrial energy metabolism?

ATP5I functions as part of the ATP synthase complex (Complex V) within the mitochondria. This small protein (7.9 kDa, 69 amino acids) is a component of the F0 domain, the membrane-embedded portion of the ATP synthase that contains the proton channel . During oxidative phosphorylation, ATP5I helps maintain the stability of F1F0-ATP synthase dimers, which is crucial for proper cristae morphology . The protein is primarily located with subunit a in the mitochondrial inner membrane and participates in the coupling mechanism whereby proton translocation through F0 drives ATP synthesis in the F1 catalytic domain via a rotary mechanism of the central stalk subunits .

Recent research has revealed that ATP5I may have additional significance beyond its structural role in ATP synthase. It has been identified as a potential target of medicinal biguanides and may play an important role in cancer cell metabolism, particularly in pancreatic cancer .

Detecting ATP5I via Western blot requires specific optimization due to its small size (7.9 kDa):

Recommended Protocol Modifications:

• Gel selection: Use high percentage (15-20%) SDS-PAGE gels or gradient gels (4-20%) to properly resolve small proteins

• Transfer conditions: Employ semi-dry transfer with reduced methanol concentration (10%) or wet transfer at lower voltage for longer time to prevent protein loss

• Membrane selection: PVDF membranes with 0.2 μm pore size rather than 0.45 μm to better capture small proteins

• Blocking optimization: Use 3-5% BSA in TBS-T rather than milk to reduce background

• Antibody dilutions: Begin with 1:500-1:1000 for primary ATP5I antibodies, as recommended by multiple suppliers

• Detection systems: High-sensitivity chemiluminescence or fluorescence detection systems

When analyzing Western blot results, researchers should be aware that ATP5I appears at approximately 8 kDa as observed across multiple validation studies . Variations in apparent molecular weight may occur due to post-translational modifications or differences in gel systems.

For IHC Applications:

• Antigen retrieval: Use TE buffer pH 9.0 as suggested by Proteintech, though citrate buffer pH 6.0 may serve as an alternative

• Antibody dilutions: Begin with 1:50-1:200 for IHC applications

• Detection systems: Both DAB and AP chromogens have been successfully used

• Controls: Include positive controls such as liver tissue, which shows consistent ATP5I expression

For IF/ICC Applications:

• Fixation methods: 4% paraformaldehyde for 15-20 minutes at room temperature preserves mitochondrial morphology

• Permeabilization: 0.1-0.3% Triton X-100 for sufficient mitochondrial access

• Co-staining: Consider dual labeling with other mitochondrial markers (e.g., TOMM20, COX IV) for colocalization studies

• Fluorophore options: Both directly conjugated antibodies (e.g., CoraLite® Plus 488) and secondary antibody detection methods work well

Researchers should note that ATP5I demonstrates a characteristic mitochondrial staining pattern in both IHC and IF applications, appearing as punctate or reticular structures consistent with mitochondrial localization .

How can researchers verify ATP5I antibody specificity in their experimental systems?

Ensuring antibody specificity is critical for reliable ATP5I detection. Several validation approaches are recommended:

• Positive and negative controls:

  • Validated positive samples: HepG2 cells, mouse liver tissue

  • Negative controls: CRISPR-Cas9 knockout cells (as used in recent ATP5I research)

  • Competing peptide blocking experiments

• Multiple detection methods:

  • Concordance between different techniques (WB, IHC, IF)

  • Mass spectrometry validation of immunoprecipitated proteins

• Signal validation tests:

  • Antibody titration to determine optimal concentration

  • Pre-adsorption with immunizing peptide (when available)

  • RNA interference to confirm signal reduction with decreased target expression

• Cross-reactivity assessment:

  • Testing in multiple species where sequence homology is known

  • Evaluating potential cross-reactivity with closely related proteins

Several commercial antibodies report extensive validation using these approaches. For instance, Proteintech antibody 16483-1-AP has been validated in multiple applications and cited in numerous publications .

What is the significance of ATP5I in cancer research and what methodologies are being used to study it?

Recent research has identified ATP5I as a potential therapeutic target in cancer, particularly through its interaction with biguanides like metformin and phenformin . Key methodologies for studying ATP5I in cancer include:

• Genetic manipulation approaches:

  • CRISPR-Cas9 knockout of ATP5I in cancer cell lines to study phenotypic effects

  • siRNA or shRNA knockdown for transient suppression

  • Rescue experiments involving reintroduction of ATP5I into knockout cells

• Metabolic analysis techniques:

  • Seahorse XF analyzer for measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

  • Lactate production assays to assess glycolytic compensation

  • ATP production assays to quantify bioenergetic effects

• Mitochondrial morphology assessment:

  • Electron microscopy for cristae structure analysis

  • Live-cell imaging with mitochondrial dyes or fluorescent proteins

  • Super-resolution microscopy for detailed structural studies

• Drug interaction studies:

  • In vitro binding assays between ATP5I and biguanide compounds

  • Dose-response studies in wildtype versus ATP5I-deficient cells

  • Combinatorial drug screening for synergistic effects

Research has shown that ATP5I knockout in pancreatic cancer cells leads to decreased levels of some respiratory complex subunits, mitochondrial morphology alterations, inhibition of oxidative phosphorylation, and increased glycolysis . Notably, ATP5I-deficient cells display resistance to the antiproliferative effects of biguanides, suggesting its role as a significant antineoplastic mitochondrial target .

What are the challenges in detecting ATP5I given its small size and mitochondrial localization?

Researchers face several technical challenges when working with ATP5I:

• Size-related detection issues:

  • The 7.9 kDa size makes protein separation and retention during experimental procedures difficult

  • Standard Western blot protocols may need significant modification

  • Risk of protein loss during membrane transfer

• Subcellular fractionation considerations:

  • Need for specialized protocols to isolate intact mitochondria

  • Potential contamination with other cellular components

  • Preservation of protein complexes during isolation

• Antibody accessibility challenges:

  • Embedded nature in the mitochondrial membrane may limit epitope exposure

  • Fixation protocols can affect antibody penetration and antigen recognition

  • Detergent selection critically impacts both solubilization and epitope preservation

• Expression level variability:

  • Tissue-specific expression differences require careful sample selection

  • Experimental conditions may alter expression levels

  • Need for sensitive detection methods due to potentially low abundance

To address these challenges, researchers should consider:

• Optimized protocols specifically designed for small mitochondrial proteins

• Validation with multiple antibodies targeting different epitopes

• Using complementary techniques beyond antibody-based detection

How can protein-protein interaction studies involving ATP5I be effectively designed and implemented?

Understanding ATP5I's interactions with other proteins, particularly within the ATP synthase complex, requires specialized approaches:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use mild detergents (e.g., digitonin 1%, DDM 1%) to preserve protein complexes

  • ATP5I antibodies have been validated for IP applications at dilutions of 1:50-1:100

  • Consider crosslinking approaches to stabilize transient interactions

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proximal proteins in living cells

  • Targeted to mitochondria via appropriate targeting sequences

  • Mass spectrometry analysis of labeled proteins

• Fluorescence-based interaction studies:

  • Förster Resonance Energy Transfer (FRET) for direct interaction assessment

  • Split-GFP complementation to visualize protein associations

  • Fluorescence Lifetime Imaging Microscopy (FLIM) for quantitative interaction analysis

• Structural biology approaches:

  • Cryo-electron microscopy of intact ATP synthase complexes

  • Cross-linking mass spectrometry (XL-MS) to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify interaction domains

When designing these experiments, researchers should be aware that ATP5I is known to interact with other subunits of the ATP synthase complex, particularly in maintaining dimer stability which affects cristae morphology .

What controls should be included when studying ATP5I in the context of mitochondrial function?

Proper controls are essential for accurate interpretation of ATP5I studies:

• Genetic controls:

  • Wildtype cells/tissues alongside ATP5I-altered samples

  • Isogenic cell lines differing only in ATP5I expression

  • Rescue experiments with re-introduction of ATP5I

• Experimental condition controls:

  • Mitochondrial function modifiers (e.g., FCCP, oligomycin, rotenone) to validate assay functionality

  • Time course experiments to distinguish acute versus chronic effects

  • Oxygen tension controls for normoxia versus hypoxia experiments

• Technical controls:

  • Multiple antibody validation including pre-immune serum controls

  • Loading controls appropriate for mitochondrial proteins (e.g., VDAC, COX IV)

  • Multiple detection methods to confirm observations

• Specificity controls:

  • Other ATP synthase subunits to distinguish ATP5I-specific effects from general complex destabilization

  • Related mitochondrial complexes to assess respiratory chain-wide effects

  • Metabolic pathway controls to identify compensatory mechanisms

A comprehensive control strategy enables researchers to distinguish direct ATP5I-mediated effects from secondary consequences or technical artifacts.

How does ATP5I contribute to mitochondrial cristae morphology and what techniques can visualize this relationship?

Recent research has established that ATP5I plays an important role in maintaining ATP synthase dimer stability, which is crucial for proper cristae morphology . To investigate this relationship:

• Electron microscopy techniques:

  • Transmission electron microscopy (TEM) for high-resolution cristae visualization

  • Electron tomography for 3D reconstruction of mitochondrial ultrastructure

  • Immuno-electron microscopy to correlate ATP5I localization with structural features

• Super-resolution fluorescence microscopy:

  • Stimulated emission depletion (STED) microscopy

  • Structured illumination microscopy (SIM)

  • Single-molecule localization microscopy (PALM/STORM)

• Live-cell imaging approaches:

  • Mitochondrial dyes (e.g., MitoTracker) combined with fluorescently tagged ATP5I

  • Time-lapse imaging to monitor dynamic changes in cristae

  • Correlative light and electron microscopy (CLEM) for integrated analysis

• Biochemical assessment of ATP synthase dimers:

  • Blue native PAGE to separate intact complexes

  • Crosslinking approaches to stabilize dimeric structures

  • Mass spectrometry to assess complex composition

These techniques have revealed that ATP5I knockout or inhibition can lead to alterations in mitochondrial morphology similar to those observed with biguanide treatment, including changes in cristae structure that affect respiratory efficiency .

What are the current hypotheses regarding ATP5I's role in mediating biguanide effects in cancer cells?

The identification of ATP5I as a target of medicinal biguanides has opened new research directions :

• Direct interaction mechanism:

  • In vitro evidence shows ATP5I can interact with biguanide analogues

  • This interaction may disrupt ATP synthase function directly

  • Structural studies are needed to elucidate the precise binding interface

• ATP synthase destabilization hypothesis:

  • Biguanide binding to ATP5I may disrupt dimer stability

  • This disruption alters cristae morphology

  • Changes in respiratory chain organization follow, affecting OXPHOS efficiency

• Metabolic reprogramming consequences:

  • Inhibition of oxidative phosphorylation via ATP5I leads to compensatory glycolysis

  • This metabolic shift may be particularly detrimental to certain cancer types

  • Therapeutic window may exist between cancer and normal cells

• Anti-proliferative mechanism:

  • ATP5I knockout cells show resistance to biguanide anti-proliferative effects

  • Reintroduction of ATP5I restores biguanide sensitivity

  • This suggests ATP5I is necessary for biguanide-mediated growth inhibition

These findings suggest ATP5I represents a significant antineoplastic mitochondrial target of biguanides like metformin and phenformin, with potential implications for developing more targeted mitochondrial cancer therapies .

To comprehensively assess how ATP5I affects mitochondrial energy production:

• Respirometry techniques:

  • Seahorse XF analyzer for real-time OCR and ECAR measurements

  • High-resolution respirometry (Oroboros) for detailed respiratory parameters

  • Clark-type oxygen electrodes for isolated mitochondria studies

• ATP production assays:

  • Luminescence-based ATP determination

  • 31P-NMR spectroscopy for in vivo ATP dynamics

  • ATP/ADP ratio measurements using fluorescent sensors

• Membrane potential assessment:

  • Potentiometric dyes (TMRM, JC-1)

  • Flow cytometry for population analysis

  • Live-cell microscopy for single-cell dynamics

• Enzyme activity measurements:

  • ATP synthase activity assays (spectrophotometric)

  • In-gel activity assays following blue native PAGE

  • Respiratory chain complex activities to assess broader impact

• Metabolic flux analysis:

  • 13C-labeled substrate tracing

  • Mass spectrometry metabolomics

  • NMR-based metabolomics for pathway analysis