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Figure 1 (A) A protein doublet of 148/150 kDa interacts specifically with reaper (RPR) beads. Recombinant GST–reaper protein on glutathione–Sepharose beads ('RPR beads') was incubated with Xenopus egg extract for 1 h at 4°C. The beads were pelleted, washed twice with egg lysis buffer (ELB), twice with NaBicarb buffer, resuspended in NaBicarb and incubated with a succinimide ester of biotin for 1 h at room temperature. The beads were then pelleted, washed twice with ELB, resuspended in SDS sample buffer and processed for a Western blot using an HRP-linked streptavidin antibody. (B) Predicted amino acid sequence of Scythe ORF. Eleven tryptic and Lys-C peptides that were found in the two different Scythe isoforms (148 and 150 kDa) are indicated as follows: single underlining = 150 kDa; double underlining = 148 kDa; dotted underlining = 148 + 150 kDa. (C) In vitro transcribed/translated Scythe protein interacts with reaper. 35S-Labeled Scythe protein was incubated with GST or GST–reaper beads in the presence of either heat-inactivated FBS or Xenopus egg extract for 30 min at room temperature. Beads were then washed three times with ELB, resolved by SDS–PAGE and processed for autoradiography.
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 | Figure 2 ScytheC312 induces morphological characteristics of apoptosis and DEVDase activation. The indicated GST fusion proteins (final concentration 600 ng/ l) or an equivalent amount of GST protein alone were added to Xenopus egg extract in the presence of sperm chromatin to form synthetic nuclei in the extracts (1000 nuclei/l) and an ATP regeneration system. (A) Photomicrographs of representative nuclei upon staining with the DNA intercalating dye, Hoescht 33258, 90 min after protein addition: dotted arrow = uncondensed interphase chromatin contained within an intact nuclear envelope, solid arrow = condensed, apoptotically fragmented chromatin, * = background staining of membranes present in the extract. (B) At the indicated times, 2 l of extract were collected for a DEVD-pNA cleavage assay. (C) The indicated amounts of ScytheC312 were added to extracts and, at the indicated times, 2 l of extract were collected for a DEVD-pNA cleavage assay.
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Figure 3 ScytheC312, but not ScytheN435 or C235, interacts with reaper. 35S-Labeled full-length (F.L.) Scythe, ScytheC312, C235 and N435 proteins were incubated with GST or GST–reaper beads in the presence of Xenopus egg extract for 60 min at room temperature. Beads were then washed three times with ELB, resolved by SDS–PAGE and processed for autoradiography. If 100% of the input 35S-labeled proteins were bound to reaper, the intensity of the signal would be equivalent to that seen in the control 'Load' lanes. Note that 100% recovery is unlikely due to competition from endogenous Scythe present in the extract.
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 | Figure 4 ScytheC312 accelerates cytochrome c release from mitochondria. Recombinant GST protein or the indicated GST fusion proteins were added to either (A) crude egg extract, (B) mitochondria and egg cytosol, or (C) buffer alone. At the indicated times, the samples were filtered through a 0.1 M microfilter to remove particulate components, including mitochondria. Aliquots (10 l) of protein filtrate were separated by SDS–PAGE and processed for Western blot with an anti-cytochrome c monoclonal antibody. Note that there is extract to extract variability in the absolute timing of cytochrome c release. The cytosol in (B) is not from the same batch of Xenopus eggs as the crude extract used in (A). Hence, the absolute time course of cytochrome c release is slightly different in these two panels.
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Figure 5 Depletion of Scythe inhibits reaper-induced DEVDase activation and mitochondrial cytochrome c release. (A) Scythe was immunodepleted from 100 l of crude extract using anti-ScytheC312 sera linked to protein A–Sepharose. After three successive rounds of immunodepletion, 10 l aliquots of extract were resolved by SDS–PAGE and processed for immunoblotting using anti-peptide sera targeted against the C-terminal 40 amino acids of the Xenopus Scythe protein. Lane 1, undepleted extract; lane 2, extract depleted with anti-Scythe C312 sera; lane 3, extract depleted with pre-immune sera. (B) Recombinant reaper protein (600 ng/l) was added to either extract depleted of endogenous Scythe protein or extracts similarly treated with pre-immune sera. At the indicated times, 2 l aliquots of extract were processed for DEVD-pNA cleavage activity. (C) Recombinant reaper protein (600 ng/l) was added to either Scythe-depleted or pre-immune-depleted Xenopus egg extracts. At the indicated times, the samples were filtered through a 0.1 M microfilter to remove particulate components, including mitochondria. Aliquots (10 l) of cytosolic protein were separated by SDS–PAGE and processed for Western blot with an anti-cytochrome c monoclonal antibody. (D) Recombinant, active caspase 8 (lacking the pro-domain; 400 ng/l) was added to buffer (no extract), extract depleted of endogenous Scythe protein or extracts similarly treated with pre-immune sera. At the indicated times, 2 l aliquots of extract were processed for DEVD-pNA cleavage activity.
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 | Figure 6 The C-terminal 312 amino acids of Scythe are capable of inducing DEVDase activation in extracts depleted of endogenous Scythe protein. Recombinant reaper protein (600 ng/l), an equivalent amount of recombinant ScytheC312 protein, or buffer was added to either Scythe-depleted or pre-immune-depleted extracts and, at the indicated times, 2 l aliquots of extract were processed for DEVD-pNA cleavage activity.
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Figure 7 Depletion of ScytheC312-interacting factors inhibits reaper-induced DEVDase activation. Recombinant reaper was added to Xenopus egg extract that had been depleted with the indicated 'beads'. At the indicated times, 2 l aliquots of extract were collected for a DEVD-pNA cleavage assay.
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