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

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. Synopsis Dedicated proteins are known to control the architecture of the inner mitochondrial membrane; however, the role of specific lipids is less well defined. Here, a combination of methods including tomography and lipidomics is used to characterise the role of lipid composition in defining cristae architecture. As lipid ratios change, a critical breakpoint phenocopies the loss of cristae‐shaping proteins in the IMM of yeast mitochondria. Phospholipid saturation controls membrane mechanical properties and modulates ATP synthase oligomerization. The mitochondria‐specific lipid cardiolipin functionally compensates for increased phospholipid saturation and is required for cristae formation in low‐oxygen environments. A mathematical model for cristae membrane tubules predicts a snap‐through instability mediated by both protein and lipid‐encoded curvatures. Together, the amount of lipid saturation and cardiolipin define mitochondrial ultrastructure.