Abstract
Scythe (BAT3; HLA-B associated transcript 3, Bag 6) is a protein that has been implicated in apoptosis because it can modulate the Drosophila melanogaster apoptotic regulator, Reaper. Mice lacking Scythe show pronounced defects in organogenesis and in the regulation of apoptosis and proliferation during mammalian development. However, the biochemical pathways important for Scythe function are unknown. We report here multiple levels of interaction between Scythe and the apoptogenic mitochondrial intermembrane protein AIF (apoptosis-inducing factor). Scythe physically interacts with AIF and regulates its stability. AIF stability is markedly reduced in Scythe(-/-) cells, which are more resistant to endoplasmic reticulum stress induced by thapsigargin. Reintroduction of Scythe or overexpression of AIF in Scythe(-/-) cells restores their sensitivity to apoptosis. Together, these data implicate Scythe as a regulator of AIF.
Highlights
Resulted in lethality associated with pronounced developmental defects in the lung, kidney, and brain [6]
ScytheϪ/Ϫ MEFs Are Resistant to Thapsigargin-induced Cell Death—We previously found that ScytheϪ/Ϫ primary cortical mine the specificity of this interaction and if it involves the ubiquitin-like N-terminal domain of Scythe, we tested the interaction between Scythe and RAD23, which contains a ubiquitin-like domain, and found that RAD23, even when neurons were more resistant than WT cells to cell death result- highly expressed, does not interact with apoptosis-inducing factor (AIF)
Ing from endoplasmic reticulum stress signaling induced by TG This suggests that the interaction between AIF and Scythe is or menadione [6]
Summary
Cells and Culture Conditions—Human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 4 mM glutamine, and 100 units of penicillin and streptomycin in a 10% CO2 humidified incubator. Lysates were immunoprecipitated overnight at 4 °C with 5 g of the indicated antibodies, followed by a 2-h incubation at 4 °C with Protein A- or G-Sepharose beads and washed three times in lysis buffer. For cells transfected with GFP-AIF or Scythe fusion proteins, cells were incubated for 30 min at 37 °C with 100 nM of a red fluorescent dye that stains the mitochondria in live cells (MitoTracker Red CMXRos; Molecular Probes), loading solution was replaced with prewarmed medium and cells were directly observed using a confocal microscope (Leica TCS Sp2). Immune complexes recovered by protein A-Sepharose and washed three times with lysis buffer were denatured and electrophoretically separated on NOVEX 8% Tris glycine denaturing gels (Invitrogen), transferred to polyvinylidene difluoride membranes. Several time points and concentrations of TG were tested using either
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