Sphingolipid Metabolism Cooperates with BAK and BAX to Promote the Mitochondrial Pathway of Apoptosis

Abstract

SUMMARY Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO4 and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions

The Gβγ-Src signaling pathway regulates TNF-induced necroptosis via control of necrosome translocation

“…The heterotypic membrane fraction was shown to play an important role in apoptosis [71]. Crudely prepared mitochondria were highly sensitive to BID-induced cytochrome C release, while highly purified mitochondria were less sensitive.…”

“…We further fractionated the HM fraction into pure mitochondrial and heterotypic membrane fractions using a published method [71,72]. The heterotypic membrane fraction is also called microsome (µS) [71]. The transmission electron microscopy pictures of mitochondrial and heterotypic membrane fractions were shown in Supplementary information, Figure S6E.…”

“…Indeed, we found that TNF plus zVAD treatment led to an enrichment of RIP1, RIP3 and MLKL in the HM fraction ( Figure 7D). We further fractionated the HM fraction into pure mitochondrial and heterotypic membrane fractions using a published method [71,72]. The heterotypic membrane fraction is also called microsome (µS) [71].…”

Mitochondrial outer membrane permeabilization: a focus on the role of mitochondrial membrane structural organization

“…Moreover, formation of the mitochondrial apoptosis-induced channel (MAC) mediated by Bax/Bak has also being ruled out, since MOMP results in both small and large molecule release whereas MAC only allows limited molecule sizes to pass through (Tait and Green 2010). Bcl-2 proteins are also able to interact with ceramide channel-forming sphingolipid to induce permeabilization (Chipuk et al 2012) or by forming putative cytochrome c release channels on the MOM which is functionally redundant to Bax/Bak (Dejean et al 2005). However, it now appears that Bax/Bak oligomerization is the key step in the formation of the apoptotic pore in the membrane for the permeabilization (Garcia-Saez 2012).…”

“…Among the products of metabolism is ceramide which can form a channel in the membrane (Hannun and Obeid 2008). Sphingosine-1-PO 4 (S1P) and hexadecanal (hex) which are ceramide precursors can mediate Bax/Bak activation through their synergistic activity leading to MOMP (Chipuk et al 2012). Overall, CL The yeast mitochondrial fraction is specific to the outer mitochondrial membrane while fractionation of the rat liver, hepatoma and human heart cell involved compositional analysis of the entire mitochondria.…”