Isolation and Phospholipid Enrichment of Muscle Mitochondria and Mitoplasts

The efficient ATP production in mitochondria relies on the highly specific organization of its double membrane. Notably, the inner mitochondrial membrane (IMM) displays a massive surface extension through its folding into cristae, along which concentrate respiratory complexes and oligomers of the ATP synthase. Evidence has accumulated to highlight the importance of a specific phospholipid composition of the IMM to support mitochondrial oxidative phosphorylation. Contribution of specific phospholipids to mitochondrial ATP production is classically studied by modulating the activity of enzymes involved in their synthesis, but the interconnection of phospholipid synthesis pathways often impedes the determination of the precise role of each phospholipid. Here, we describe a protocol to specifically enrich mitochondrial membranes with cardiolipin or phosphatidylcholine, as well as a fluorescence-based method to quantify phospholipid enrichment. This method, based on the fusion of lipid vesicles with isolated mitochondria, may further allow a precise evaluation of phospholipid contribution to mitochondrial functions.

Automated summary

Efficient ATP production in mitochondria relies on the specific phospholipid composition of the inner mitochondrial membrane. Studying the contribution of specific phospholipids to mitochondrial functions is often hindered by the interconnection of phospholipid synthesis pathways, but a protocol for enriching mitochondrial membranes with specific phospholipids and quantifying the enrichment through a fluorescence-based method can help overcome this obstacle.