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Diverse changes have been described in mitochondria of apoptotic cells: the phospholipid content is modified, ceramide and GD3 concentrations increase, the cristae structure is modified, and nonresident proteins are recruited into the mitochondrial membranes. In particular, Bax, a Bcl‐2 family member protein, moves from the cytosol to the mitochondria, inducing cytochrome c release. Modifications of the content and distribution of specific lipids in the mitochondrial membranes, along with the well‐known participation of the mitochondrial permeability transition pore in triggering apoptosis, led us to propose that lipid microdomains in mitochondria could coexist as structural elements with some of the mitochondrial permeability transition pore‐forming proteins and with members of the Bcl‐2 family. In this work, we demonstrated that Bax was associated preferentially with mitochondrial detergent‐resistant membranes (mDRMs) in reperfused rat hearts, a well‐known apoptotic model. Bax insertion into mDRMs correlated with cytochrome c release from such mitochondria. Bax location in mDRMs was associated with both the voltage‐dependent anion channel and the adenine nucleotide translocator, two mitochondrial permeability transition pore‐forming proteins. Interestingly, the voltage‐dependent anion channel was more abundant in the mDRM fraction than in the Triton X‐100‐soluble fraction. Ceramide and cholesterol contents were higher in mDRMs from reperfused hearts. Our results suggest that membrane microenvironments enriched in cholesterol and ceramide in mitochondria favor Bax translocation to this organelle, fostering propagation of the apoptotic cascade. Structured digital abstract  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7233017: Vdac (uniprotkb:http://www.ebi.uniprot.org/entry/Q9Z2L0) and Bax (uniprotkb:http://www.ebi.uniprot.org/entry/Q63690) colocalize (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0403) by cosedimentation through density gradients (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0029)

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