STRUCTURE-FUNCTION COMPARISONS OF THE PROAPOPTOTIC PROTEIN BAX IN YEAST AND MAMMALIAN-CELLS

Expression of the proapoptotic protein Bax under the control of a GAL1 0 promoter in Saccharomyces cerevisiae resulted in galactose-inducible cell death. Immunofluorescence studies suggested that Bax is principa lly associated with mitochondria in yeast cells. Removal of the carbox yl-terminal transmembrane (TM) domain from Bax [creating Bax (Delta TM )] prevented targeting to mitochondrial and completely abolished cytot oxic function in yeast cells, suggesting that membrane targeting is cr ucial for Bax-mediated lethality. Fusing a TM domain from Mas70p, a ye ast mitochondrial outer membrane protein, to Bax (Delta TM) restored t argeting to mitochondria and cytotoxic function in yeast cells. Deleti on of four well-conserved amino acids (IGDE) from the BH3 domain of Ba x ablated its ability to homodimerize and completely abrogated lethali ty in yeast cells. In contrast, several Bax mutants which retained abi lity to homodimerize (Delta BH1, Delta BH2, and Delta 1-58) also retai ned at least partial lethal function in yeast cells. In coimmunoprecip itation experiments, expression of the wild-type Bax protein in Rat-1 fibroblasts and 293 epithelial cells induced apoptosis, whereas the Ba x (Delta IGDE) mutant failed to induce apoptosis and did not associate with endogenous wild-type Bax protein. In contrast to yeast cells, Ba x (Delta TM) protein retained cytotoxic function in Rat-1 and 293 cell s, was targeted largely to mitochondria, and dimerized with endogenous Bax in mammalian cells. Thus, the dimerization-mediating BH3 domain a nd targeting to mitochondrial membranes appear to be essential for the cytotoxic function of Bax in both yeast and mammalian cells.