Regulated Targeting of BAX to Mitochondria

Abstract

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, our results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting.

Bcl-xS and Bax induce different apoptotic pathways in PC12 cells

“…Following overexpression of Bcl-x S , the transfected cells die and the exogenous Bclx S is presented in the mitochondria. These results therefore suggest that, as shown previously for other pro-apoptotic members of the family [such as Bax and Bid (Goping et al, 1998;Li et al, 1998)], translocation of Bcl-x S to the mitochondria is important for the Bclx S -induced death e ect. It is not known why overexpression of Bcl-x S resulted in its translocation to the mitochondria.…”

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

“…Thus, Bak activation enhances α9 membrane insertion, as observed for a semi-cytosolic Bak mutant 33 and for Bax. 48 Bak α9 traverses the MOM but does not line a pore following apoptosis. To identify α9 residues buried in the hydrophobic interior of the MOM, cysteine variants were labeled with the membrane-impermeable sulfhydryl reagent 4-acetamido-4ʹ-((iodoacetyl)amino)stilbene-2,2ʹ-disulfonic acid (IASD).…”

Complete activation of Bax by a single site mutation

“…As for the role of Bax's N terminus in regulating the solubility of this protein, an N-terminal epitope of this protein has been shown to become exposed when Bax translocates to mitochondria. In addition, Goping et al (1998) have shown that the first 19 amino acids of Bax, or the ART sequence, is important in regulating Bax localization. Deletion of this sequence will also result in the constitutive localization of Bax to mitochondria (Cartron et al, 2003).…”