Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to 
and uses mitochondria as its major site of action. Bax normally resides in the 
cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and 
it promotes apoptosis in two ways: (i) by disrupting mitochondrial membrane 
barrier function by formation of ion-permeable pores in mitochondrial membranes 
and (ii) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain 
and inhibiting their functions. A hairpin pair of amphipathic alpha-helices 
(alpha5-alpha6) in Bax has been predicted to participate in membrane insertion 
and pore formation by Bax. We mutagenized several charged residues in the 
alpha5-alpha6 domain of Bax, changing them to alanine. These substitution 
mutants of Bax constitutively localized to mitochondria and displayed a 
gain-of-function phenotype when expressed in mammalian cells. Furthermore, 
substitution of 8 out of 10 charged residues in the alpha5-alpha6 domain of Bax 
resulted in a loss of cytotoxicity in yeast but a gain-of-function phenotype in 
mammalian cells. The enhanced function of this Bax mutant was correlated with 
increased binding to Bcl-X(L), through a BH3-independent mechanism. These 
observations reveal new functions for the alpha5-alpha6 hairpin loop of Bax: (i) 
regulation of mitochondrial targeting and (ii) modulation of binding to 
antiapoptotic Bcl-2 proteins.