The role of cytochrome c in caspase activation in Drosophila melanogaster cells

Abstract

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.

Mechanism of action of Drosophila Reaper in mammalian cells: Reaper globally inhibits protein synthesis and induces apoptosis independent of mitochondrial permeability

“…Cyt c does, however, adopt an altered conformation during the apoptotic process, also in response to Reaper expression. 34,35 Caspases were seen to cluster around the mitochondria and mitochondria from apoptotic cells could trigger effector caspase function in vitro, arguing that some active upstream component was associated with them. 34,35 Drosophila cells express a homologue of the mammalian Apaf-1 scaffold protein, which can bind Cyt c in vitro.…”

“…34,35 Caspases were seen to cluster around the mitochondria and mitochondria from apoptotic cells could trigger effector caspase function in vitro, arguing that some active upstream component was associated with them. 34,35 Drosophila cells express a homologue of the mammalian Apaf-1 scaffold protein, which can bind Cyt c in vitro. Moreover, in extracts from Drosophila cells, exogenous Cyt c can promote caspase activation, dependent on endogenous Apaf-1 expression.…”

“…The following day, equal numbers of cells were labelled with [ 35 S]-methionine/cysteine mixture for 1 h. Cell lysates were prepared and either separated by SDS-PAGE or subject to TCA precipitation. Radiolabelled proteins were detected by autoradiography or quantitated by scintillation counting equal numbers of cells from each transfected line were labelled for 1 h with 35 S methionine/cysteine and analysed for the presence of radiolabelled protein by SDS-PAGE of total cell lysates and scintillation counting of total TCA-precipitated protein derived from cell lysates. Both Reaper and DIBM Reaper efficiently reduced radiolabelled protein levels detectable by autoradiography.…”

Molecular determinants of the subcellular localization of the Drosophila Bcl-2 homologues DEBCL and BUFFY

“…5 Â 10 6 cells were lysed in Buffer A (20 mM HEPES-KOH pH 7.5, 50 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT in 250 mM sucrose supplemented with protease inhibitors) and harvested by differential centrifugation for HM LM and cytosolic fractions as previously described. 15 Cell death assays. SL2 cells were transiently transfected with 1.5 mg buffy and/or debcl expression constructs together with 0.5 mg pIE1.4-LacZ reporter construct using Cellfectin.…”

“…15 As shown in Figure 5, DEBCL is predominantly present in heavy membrane (HM) fractions comprising mitochondria. While DEBCL mutants could still localize to HM, there was a notable amount of protein in cytosolic fractions confirming our immunofluorescence data that mutation of the X domain and B domain can significantly mislocalize DEBCL protein ( Figure 5).…”