Abstract
REAPER (RPR) is a 65-amino acid protein that is critical activator of programmed cell death in Drosophila. On the basis of sequence alignment data, it was recently proposed that RPR might represent an ancestral molecule from which the death domain in a number of proteins may have evolved. We tested this idea by examining the activity of mutations in RPR that parallel inactivation mutations of the tumor necrosis factor receptor 1 death domain. The RPR mutants retained potent apoptotic function, suggesting that cell death activity mediated by RPR is distinct from signaling by the tumor necrosis factor receptor 1 death domain.
Highlights
A genomic interval spanning the reaper gene is required for programmed cell death in Drosophila [1,2,3]
Based on sequence alignments and functional arguments, it was proposed that rpr is a member of the family of death domain containing proteins [9] and might represent an ancestral cytoplasmic cell death factor [11, 12]
This is an attractive scenario consistent with the fact that apoptosis induced either by RPR or death domain proteins is associated with elevation of ceramide and requires interleukin-1-converting enzyme-like protease activity [6, 13,14,15,16,17]
Summary
Gene Constructions—pMT-rpr expresses an amino-terminal HA-tag fused to the rpr ORF from the Drosophila metallothionein promoter (referred to as pMt-HA-rpr in Ref. 4) To produce this plasmid, appropriate oligo linkers were ligated into the BsrDI site of the rpr cDNA creating pRpr-BS, thereby introducing BamHI and SalI sites just after the rpr termination codon. PMT-rpr⌬2–15 removes amino acids 2–15 of RPR from pMT-rpr and was confirmed by sequence analysis This mutation was produced by inserting a polymerase chain reaction-derived fragment into a derivative of pRMHa.. The Chameleon kit (Stratagene) was used in conjunction with a mutagenic primer to produce pMT-rprE29A from the parental pMT-rpr vector This mutation was confirmed by sequence analysis and substitutes alanine for glutamic acid 29 of RPR. Transfections—Stably transfected cell lines were produced by cotransfection with pCohygro [21] into Schneider L2 cells [22] cultured in Sf900 II SFM (Life Technologies, Inc.) medium with 50 g/ml Gentamicin (Sigma). For assays of cell loss, cells were fixed with 2% formaldehyde for 30 min, washed with phosphate-buffered saline, and stained with X-Gal to visualize cotransfected -gal activity [23]. HA-RPR and HARPR⌬2–15 proteins were visualized by immunoblotting, using anti-HA monoclonal antibody (Boehringer Manheim) as described previously [4]
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