Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components^1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca²⁺ channel are known as the mitochondria associated-ER membranes or MAMs^4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca^{2+ 4,5,6}. The ER serves as the primary store of intracellular Ca²⁺, and in this capacity regulates a myriad of cellular processes downstream of Ca²⁺ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca²⁺ homeostasis, by buffering cytosolic Ca²⁺ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca²⁺ unbalance^4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca²⁺ signaling and regulation of mitochondrial Ca²⁺ concentration, lipid biosynthesis and transport, energy metabolism and cell survival ^4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells^13,14.

Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein–protein interactions^4,15.