Purification of Functional F-ATP Synthase from Blue Native PAGE

Galber, Chiara; Valente, Giulia; Stockum, Sophia von; Giorgio, Valentina

doi:10.1007/978-1-4939-9018-4_20

Cited by 7 publications

(5 citation statements)

References 29 publications

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“…ATP synthase was immunoprecipitated from mitochondria that were maintained under different functional conditions (stimulated ATP hydrolysis, State 2 and State 3 steady state respiratory conditions). Mitochondrial extracts were obtained applying 1% (w/ v) digitonin, resulting in a mixed population of oligomeric, dimeric, and monomeric ATP synthase complexes [45,49]. IF1 was detected in mitochondrial extracts (before immunoprecipitation) and in anti-ATP synthase immunoprecipitated fractions (eluates, Fig.…”

Section: Resultsmentioning

confidence: 99%

The mitochondrial inhibitor IF1 binds to the ATP synthase OSCP subunit and protects cancer cells from apoptosis

et al. 2023

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The mitochondrial protein IF1 binds to the catalytic domain of the ATP synthase and inhibits ATP hydrolysis in ischemic tissues. Moreover, IF1 is overexpressed in many tumors and has been shown to act as a pro-oncogenic protein, although its mechanism of action is still debated. Here, we show that ATP5IF1 gene disruption in HeLa cells decreases colony formation in soft agar and tumor mass development in xenografts, underlining the role of IF1 in cancer. Notably, the lack of IF1 does not affect proliferation or oligomycin-sensitive mitochondrial respiration, but it sensitizes the cells to the opening of the permeability transition pore (PTP). Immunoprecipitation and proximity ligation analysis show that IF1 binds to the ATP synthase OSCP subunit in HeLa cells under oxidative phosphorylation conditions. The IF1–OSCP interaction is confirmed by NMR spectroscopy analysis of the recombinant soluble proteins. Overall, our results suggest that the IF1-OSCP interaction protects cancer cells from PTP-dependent apoptosis under normoxic conditions.

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Section: Resultsmentioning

confidence: 99%

The mitochondrial inhibitor IF1 binds to the ATP synthase OSCP subunit and protects cancer cells from apoptosis

et al. 2023

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“…The emergence of ATP synthase dimers and other cristae‐shaping protein complexes occurred alongside a specialization in the inner membrane lipidome, including the proliferation of CL from the proteobacterial inner membrane (Sohlenkamp & Geiger, 2016 ; Rowlett et al , 2017 ). Among extant eukaryotes, ATP synthase organization varies widely; for example, in mammalian mitochondria, ATP synthases range from primarily monomeric (Galber et al , 2019 ) to mixtures of monomers and dimers (Bisetto et al , 2007 ). It is notable that CL is essential for proper IMM structure in these systems, unlike in aerobic yeast in which dimers are predominant.…”

Section: Discussionmentioning

confidence: 99%

Cristae formation is a mechanical buckling event controlled by the inner mitochondrial membrane lipidome

Venkatraman,

Lee,

Garcia

et al. 2023

The EMBO Journal

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Cristae are high‐curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae‐shaping proteins have been defined, analogous lipid‐based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi‐scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein‐mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low‐oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM.

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“…The emergence of ATP synthase dimers and other cristae-shaping protein complexes occurred alongside a specialization in the inner membrane lipidome, including the proliferation of CL from the proteobacterial inner membrane (Sohlenkamp & Geiger, 2016; Rowlett et al , 2017). Among extant eukaryotes, ATP synthase organization varies widely; for example, in mammalian mitochondria ATP synthases range from primarily monomeric (Galber et al , 2019) to mixtures of monomers and dimers (Bisetto et al , 2007). It is notable that CL is essential for proper IMM structure in these systems, unlike in aerobic yeast in which dimers are predominant.…”

Section: Discussionmentioning

confidence: 99%

Cristae formation is a mechanical buckling event controlled by the inner membrane lipidome

Venkatraman

Lee

García

et al. 2023

Preprint

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The inner mitochondrial membrane (IMM) is the site of bulk ATP generation in cells and has a broadly conserved lipid composition enriched in unsaturated phospholipids and cardiolipin (CL). While proteins that shape the IMM and its characteristic cristae membranes (CM) have been defined, specific mechanisms by which mitochondrial lipids dictate its structure and function have yet to be elucidated. Here we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions shape CM morphology and ATP generation. When modulating fatty acid unsaturation in engineered yeast strains, we observed that loss of di-unsaturated phospholipids (PLs) led to a breakpoint in IMM topology and respiratory capacity. We found that PL unsaturation modulates the organization of ATP synthases that shape cristae ridges. Based on molecular modeling of mitochondrial-specific membrane adaptations, we hypothesized that conical lipids like CL buffer against the effects of saturation on the IMM. In cells, we discovered that loss of CL collapses the IMM at intermediate levels of PL saturation, an effect that is independent of ATP synthase oligomerization. To explain this interaction, we employed a continuum modeling approach, finding that lipid and protein-mediated curvatures are predicted to act in concert to form curved membranes in the IMM. The model highlighted a snapthrough instability in cristae tubule formation, which could drive IMM collapse upon small changes in composition. The interaction between CL and di-unsaturated PLs suggests that growth conditions that alter the fatty acid pool, such as oxygen availability, could define CL function. While loss of CL only has a minimal phenotype under standard laboratory conditions, we show that its synthesis is essential under microaerobic conditions that better mimic natural yeast fermentation. Lipid and protein-mediated mechanisms of curvature generation can thus act together to support mitochondrial architecture under changing environments.

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Purification of Functional F-ATP Synthase from Blue Native PAGE

Cited by 7 publications

References 29 publications

The mitochondrial inhibitor IF1 binds to the ATP synthase OSCP subunit and protects cancer cells from apoptosis

The mitochondrial inhibitor IF1 binds to the ATP synthase OSCP subunit and protects cancer cells from apoptosis

Cristae formation is a mechanical buckling event controlled by the inner mitochondrial membrane lipidome

Cristae formation is a mechanical buckling event controlled by the inner membrane lipidome

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