Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells.
Expression of the proapoptotic protein Bax under the control of a GAL10 promoter in Saccharomyces cerevisiae resulted in galactose-inducible cell death. Immunofluorescence studies suggested that Bax is principally associated with mitochondria in yeast cells. Removal of the carboxyl-terminal transmembrane (TM) domain from Bax [creating Bax (deltaTM)] prevented targeting to mitochondrial and completely abolished cytotoxic function in yeast cells, suggesting that membrane targeting is crucial for Bax-mediated lethality. Fusing a TM domain from Mas70p, a yeast mitochondrial outer membrane protein, to Bax (deltaTM) restored targeting to mitochondria and cytotoxic function in yeast cells. Deletion of four well-conserved amino acids (IGDE) from the BH3 domain of Bax ablated its ability to homodimerize and completely abrogated lethality in yeast cells. In contrast, several Bax mutants which retained ability to homodimerize (deltaBH1, deltaBH2, and delta1-58) also retained at least partial lethal function in yeast cells. In coimmunoprecipitation experiments, expression of the wild-type Bax protein in Rat-1 fibroblasts and 293 epithelial cells induced apoptosis, whereas the Bax (deltaIGDE) mutant failed to induce apoptosis and did not associate with endogenous wild-type Bax protein. In contrast to yeast cells, Bax (deltaTM) protein retained cytotoxic function in Rat-1 and 293 cells, was targeted largely to mitochondria, and dimerized with endogenous Bax in mammalian cells. Thus, the dimerization-mediating BH3 domain and targeting to mitochondrial membranes appear to be essential for the cytotoxic function of Bax in both yeast and mammalian cells.
The Burnham Institute, Cancer Research Center, La Jolla, California 92037, USA.
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Fisk
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Yaffe
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Mahajan
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Herman
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Reed
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008322:Mammals; D051379:Mice; D008854:Microscopy, Electron; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000502:physiology* / Q000648:ultrastructure; D014162:Transfection; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C494827:Bax protein, mouse; C494828:Bax protein, rat; D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein