Recombinant Rat ATP synthase subunit f, mitochondrial (Atp5j2)

Basic Research Questions

• What is the physiological role of ATP synthase subunit f (Atp5j2) in mitochondrial function?

ATP synthase subunit f (Atp5j2) is an essential component of the mitochondrial ATP synthase complex (F₀F₁ complex), which plays a crucial role in cellular energy production. Located in the inner mitochondrial membrane, this subunit contributes to the formation of the membrane integral F₀ domain of ATP synthase. The ATP synthase complex generates ATP from ADP using the proton gradient across the inner mitochondrial membrane, which is established by the electron transport chain .

Atp5j2 is part of the membrane domain that facilitates proton translocation through the F₀ portion, enabling the rotation of the c-ring that ultimately drives ATP synthesis in the catalytic F₁ domain. This is critical for maintaining cellular energy homeostasis in energy-demanding tissues like cardiac and skeletal muscle .

• How is recombinant Atp5j2 typically expressed and purified for research applications?

Recombinant Rat ATP synthase subunit f (Atp5j2) can be expressed using several expression systems, with the following methodological considerations:

The choice of expression system depends on experimental requirements. For structural studies requiring high purity, in vitro cell-free expression systems can achieve >90% purity as determined by SDS-PAGE . Bacterial expression is often sufficient for producing antigens for antibody production, while mammalian expression may be preferred for functional studies .

• What validation methods are recommended to confirm the identity and activity of recombinant Atp5j2?

Validation of recombinant Atp5j2 should include multiple complementary approaches:

• Molecular confirmation: Western blotting with anti-GFP or specific anti-ATP5J2 antibodies when using fusion proteins (e.g., ATP5B-paGFP) .

• Subcellular localization: Co-localization studies using MitoTracker Red to confirm mitochondrial targeting in transfected cells .

• Protein-protein interaction: Co-immunoprecipitation to verify assembly into the ATP synthase complex .

• Functional assessment: Membrane potential measurements using fluorescent probes to assess incorporation into functional ATP synthase.

• Spectroscopic analysis: Circular dichroism to confirm proper folding of the recombinant protein.

Research by Huang et al. demonstrated that antibody-based detection, coupled with functional assays, can effectively validate the identity and activity of ATP synthase components in experimental models .

Advanced Research Questions

• How can recombinant Atp5j2 be used to study the assembly pathway of the ATP synthase membrane domain?

Recombinant Atp5j2 serves as a powerful tool for investigating ATP synthase assembly through several advanced approaches:

• Fluorescently tagged constructs: Engineering ATP5J2-paGFP (photoactivatable GFP) fusion proteins allows real-time tracking of ATP synthase subunit trafficking within live cells. This technique revealed that ATP synthase components move from the cytosolic region to the plasma membrane, providing insights into assembly dynamics .

• Assembly intermediate analysis: By introducing recombinant Atp5j2 into cells with knockouts of specific ATP synthase subunits, researchers can characterize vestigial complexes that form, revealing the step-by-step assembly process of the membrane domain .

• Cross-linking studies: Chemical cross-linking of recombinant Atp5j2 with other subunits helps map protein-protein interactions during complex formation, identifying critical interfaces for assembly.

Research has shown that ATP synthase assembly follows a specific pathway where nuclear-encoded components (including Atp5j2) form an intermediate complex that serves as a template for the insertion of mitochondrially-encoded subunits (ATP6 and ATP8) . This approach has been crucial for understanding how this complex molecular machine is built.

• What role does ATP5J2 play in mitochondrial membrane organization and potential disease mechanisms?

ATP5J2 has significant implications for mitochondrial membrane architecture and disease pathology:

Studies using recombinant ATP5J2 have revealed that this subunit contributes to the formation of ATP synthase dimers, which are critical for cristae formation and mitochondrial membrane curvature. Time-series live-cell imaging with fluorescently labeled ATP synthase components showed that these complexes can localize to the plasma membrane in certain cancer cells, forming ectopic ATP synthase (eATP synthase) .

The role of ATP5J2 in disease mechanisms includes:

• Cancer progression: eATP synthase containing ATP5J2 facilitates ATP generation in the extracellular environment, creating favorable conditions for tumor growth .

• Mitochondrial disorders: Mutations in ATP5J2 have been associated with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and Leber's hereditary optic neuropathy (LHON) .

• Cardiac dysfunction: Proteomic profiling revealed that ATP5J2 abundance increases 1.41-15.94 fold during surgical myocardial ischemia-reperfusion, suggesting a role in the cardiac stress response .

These findings indicate that ATP5J2 functions extend beyond structural roles in ATP synthase to influence membrane organization and potential pathological processes.

• How can spatial proteomics approaches be combined with recombinant Atp5j2 to study its trafficking in different cell types?

Integrating spatial proteomics with recombinant Atp5j2 creates powerful experimental paradigms:

• BioID proximity labeling: Fusion of Atp5j2 with a promiscuous biotin ligase (BirA*) allows identification of proximal proteins during trafficking, revealing dynamic interaction networks.

• APEX2 electron microscopy: APEX2-tagged Atp5j2 enables visualization of subcellular localization at ultrastructural resolution while maintaining functionality.

• Super-resolution microscopy: Techniques like PALM/STORM with tagged Atp5j2 provide nanoscale resolution of trafficking pathways.

• Live-cell imaging workflows:

  • Photoactivation of paGFP-tagged Atp5j2 allows specific tracking of protein pools

  • Images can be captured every 0.4 seconds for up to 15 minutes

  • Dual-labeling with MitoTracker Red verifies mitochondrial localization (~98% colocalization)

  • TIRF microscopy can validate mitochondria-plasma membrane fusion events

Research demonstrated that ATP synthase subunits, including Atp5j2, are assembled in mitochondria and subsequently transported along microtubules through interactions with dynamin-related protein 1 (DRP1) and kinesin family member 5B (KIF5B), ultimately reaching the cell surface .

• What are the current challenges in studying cell type-specific variation in Atp5j2 expression and function?

Research into cell type-specific variations in Atp5j2 faces several methodological challenges:

• Statistical power limitations: Studies of cell type-specific variance require large sample sizes. Analysis shows that at least 100 samples are needed to achieve >70% true positive rates when using restricted maximum likelihood (REML) methods .

• Cell type proportion effects: When analyzing heterogeneous tissues, variations in cell type proportions significantly impact the detection of cell-specific effects. Power increases when the main cell type of interest becomes more common .

• Uncertainty in expression estimates: Bootstrap resampling of cells shows that coefficient of variation in expression estimates can reach ~0.2, affecting model accuracy. The Haseman-Elston regression method remains stable under noise but has limited power compared to REML jackknife approaches .

• Technical considerations:

  • Antibody cross-reactivity between ATP5J2 and related subunits

  • Limited availability of cell type-specific promoters for targeted expression

  • Challenges in maintaining physiological expression levels in recombinant systems

To address these challenges, researchers can employ single-cell RNA sequencing, multiplexed immunofluorescence imaging, and CRISPR-mediated tagging of endogenous Atp5j2 for more precise quantification of expression variation across cell types.

• How can recombinant Atp5j2 be utilized to investigate the relationship between mitochondrial dynamics and ATP synthase function?

Recombinant Atp5j2 provides valuable tools for exploring mitochondrial dynamics:

• Fusion protein tracking: ATP5J2-paGFP constructs allow visualization of mitochondrial movement and membrane fusion events in real-time. Research has shown that ATP synthase components, including Atp5j2, move toward the plasma membrane with mitochondria, suggesting coordinated trafficking .

• Multi-color imaging approaches: Simultaneous labeling of mitochondrial outer membrane (with MOM-GFP), inner membrane (with Mito-GFP), and plasma membrane (with CellMask Deep Red) revealed that entire mitochondria containing both membranes are transported toward the cell surface .

• Experimental workflow for fusion event detection:

  • Transfection of cells with ATP5J2-paGFP constructs

  • Mitochondrial labeling with MitoTracker Red

  • Time-lapse imaging at 0.4-second intervals for up to 15 minutes

  • TIRF microscopy to detect fusion events at the basal cell surface

This approach revealed that ATP synthase is assembled in mitochondria, transported along microtubules, and delivered to the cell surface through a process where mitochondrial membranes fuse with the plasma membrane . This mechanism provides insight into how ATP synthase components, including Atp5j2, contribute to mitochondrial dynamics.

• What are the implications of the ATP5MF-PTCD1 readthrough transcription for experimental design when studying Atp5j2?

The discovery of ATP5MF-PTCD1 readthrough transcription presents significant experimental design considerations:

The ATP5MF-PTCD1 locus represents a naturally occurring readthrough transcription between the ATP5J2 (ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2) and PTCD1 (pentatricopeptide repeat domain 1) genes on chromosome 7. This readthrough produces a fusion protein that shares sequence identity with both individual gene products .

Experimental design implications include:

• Primer and probe design: Researchers must carefully design PCR primers that specifically distinguish between ATP5J2 mRNA and the readthrough transcript to avoid misinterpretation of expression data.

• Antibody selection: When using antibodies for detection, epitopes should be chosen that differentiate between the individual ATP5J2 protein and the fusion protein.

• Expression construct considerations: For recombinant expression, researchers should be aware that natural readthrough occurs and may choose to include or exclude the readthrough region depending on experimental goals.

• Functional studies: Experiments should consider the potential regulatory interplay between ATP5J2 and PTCD1, as the readthrough may represent a mechanism of coordinated expression of these functionally related mitochondrial proteins.

• Disease associations: Since ATP5MF-PTCD1 is associated with congenital anomalies of kidney and urinary tract syndrome , phenotypic analyses should consider both ATP synthase function and broader developmental contexts.

• How do different expression systems affect the post-translational modifications and functional properties of recombinant Atp5j2?

Expression system selection significantly impacts recombinant Atp5j2 properties:

Research considerations:

• E. coli-expressed Atp5j2 may require refolding protocols to achieve proper structure

• Mammalian expression provides better incorporation into ATP synthase complexes

• Cell-free systems offer rapid production but may require additional optimization for functional studies