Functional genetic screening reveals the role of mitochondrial cytochrome b as a mediator of FAS-induced apoptosis.
Functional selection of genetic suppressor elements (GSEs), engineered gene fragments that interfere with the function of a particular gene product, was used to identify regulators of FAS-induced apoptosis. Chicken DF-1 cells expressing human FAS receptor and susceptible to FAS-induced apoptosis were infected with a GSE library consisting of randomly fragmented normalized chicken cDNAs in a replication-competent avian retroviral vector. Virus-producing cells were subjected to several rounds of selection using FAS agonistic antibodies, resulting in isolation of a set of GSEs conferring resistance to FAS-induced apoptosis. Surprisingly, one of the isolated GSEs encoded a 42 amino acid-long polypeptide derived from the C-terminal half of cytochrome b (Cyt b) encoded by the mitochondrial genome. Subsequent experiments showed that caspase 8-dependent cleavage of mitochondrial Cyt b and translocation of its C-terminal half into the cytoplasm occurred during FAS-induced apoptosis in both chicken and human cells. Ectopic cytoplasmic expression of either full-length Cyt b or its C-terminal half in several human cell lines induced apoptosis, which could be suppressed by the isolated GSE, but not by Bcl2 over-expression or Apaf-1 or cytochrome c knock-down. These results reveal a cytochrome c-independent branch of FAS-induced apoptosis involving cleavage and cytoplasmic release of mitochondrial Cyt b.
Komarov
Andrei P
AP
Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Rokhlin
Oskar W
OW
Yu
Chang-An
CA
Gudkov
Andrei V
AV
D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D045303:Cytochromes b / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013489:Suppression, Genetic; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
D051023:BH3 Interacting Domain Death Agonist Protein; C491901:FAS protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D019014:fas Receptor; D045304:Cytochromes c; D045303:Cytochromes b; D053181:Caspase 8
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