No abstract

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)

No abstract

Recent evidence suggests the existence of lipid microdomains in mitochondria, apparently coexisting as structural elements with some of the mitochondrial permeability transition pore‐forming proteins and members of the Bcl‐2 family. The aim of this study was to investigate the relevance of the main components of membrane microdomains (e.g. cholesterol and sphingolipids) in activation of the mitochondrial permeability transition pore (mPTP) by recombinant BAX (rBAX). For this purpose, we used chemically modified renal cortex mitochondria and renal cortex mitochondria from hypothyroid rats that show a modified mitochondrial lipid composition in vivo. Oligomeric rBAX induced an enhanced permeability conformation in the mPTP of control mitochondria. rBAX failed to induce mPTP opening when the cholesterol and ganglioside content of mitochondria were modified with the chelator methyl‐beta‐cyclodextrin. Accordingly, hypothyroid mitochondria, with endogenously lower cholesterol and ganglioside content, showed resistance to mPTP opening induced by rBAX. These observations suggest that enriched cholesterol and ganglioside domains in the mitochondrial membranes may determine BAX interaction with the mPTP. An intriguing observation was that chemical extraction of cholesterol and ganglioside in control mitochondria did not have an effect on rBAX insertion. Conversely, in hypothyroid mitochondria, rBAX insertion was diminished dramatically compared with control mitochondria. The membrane and protein changes associated with thyroid status and their possible role in rBAX docking into the membranes are discussed.

Bax, a pro-apoptotic member of the Bcl-2 family of proteins has the ability to form transmembrane pores large enough to allow cytochrome c (Cyt c) release, as well as to activate the mitochondrial permeability transition pore (mPTP); however, no differential study has been conducted to clarify which one of these mechanisms predominates over the other in the same system. In the present study, we treated isolated mitochondria from MCF7 cells with recombinant protein Bax and tested the efficacy of the mPTP inhibitor cyclosporin A (CsA) and of the Bax channel blocker (Bcb) to inhibit cytochrome c release. We also, induced apoptosis in MCF7 cell cultures with TNF-α plus cycloheximide to determine the effect of such compounds in apoptosis induction via mPTP or Bax oligomerization. Cytochrome c release was totally prevented by CsA and partially by Bcb when apoptosis was induced with recombinant Bax in isolated mitochondria from MCF7 cells. CsA increased the number of living cells in cell culture, as compared with the effect of Bax channel blocker. These results indicate that mPTP activation is the predominant pathway for Bax-induced cytochrome c release from MCF7 mitochondria and for apoptosis induction in the whole cell.