The N-terminal End of Bax Contains a Mitochondrial-targeting Signal

Abstract

The translocation of Bax ␣, a pro-apoptotic member of the BCL-2 family from the cytosol to mitochondria, is a central event of the apoptotic program. We report here that the N-terminal (NT) end of Bax ␣, which contains its first ␣ helix (⌯␣1), is a functional mitochondrial-addressing signal both in mammals and in yeast. Similar results were obtained with a newly described variant of Bax called Bax , which lacks the first 20 amino acids of Bax ␣ and is constitutively associated with mitochondria. Deletion of ⌯␣1 impairs the binding of Bax to mitochondria, whereas a fusion of the N terminus of Bax ␣, which contains ⌯␣1 with a cytosolic protein, results in the binding of the chimeric proteins to mitochondria both in a cell-free assay and in vitro. More importantly, the mitochondria-bound chimeric proteins inhibit the interaction of Bax with mitochondria as well as Baxapoptogenic properties. The mutations of the ⌯␣1, which inhibit Bax ␣ and Bax translocation to mitochondria, also block the subsequent activation of the execution phase of apoptosis. Conversely, a deletion of the C terminus does not appear to influence Bax ␣ and Bax mitochondrial addressing. Taken together, our results suggest that Bax is targeted to mitochondria by its NT and thus through a pathway that is unique for a member of the BCL-2 family.Apoptosis is finely regulated by intracellular events, which at its onset, appear to be under the control of members of the BCL-2 family (1). The main site of action of these proteins appears to be the mitochondrion, particularly through the control of the release of apoptogenic factors from this organelle (2, 3). Members of the BCL-2 family can be anti-apoptotic or proapoptotic and totally or partially mitochondrial-bound or cytosolic (4). In most cells, one of the crucial and most regulated steps in the implementation of apoptosis is the control of the translocation of Bax ␣ from the cytosol to the mitochondria (4). Anti-apoptotic proteins such as Bcl-2 or Bcl-XL act as inhibitors of Bax function, whereas proapoptotic BH3 only members can either hinder this inhibition (e. g. Bad) or directly activate Bax ␣ (e. g. Bid), which in both cases would promote the association of Bax ␣ with the mitochondrial outer membrane (MOM) 1 (5, 6). Bcl-2 is anchored into MOM bilayer by a short hydrophobic domain close to the C terminus (CT) with its N terminus (NT) exposed toward the cytosol (7-9). Bcl-2 belongs to a class of membrane proteins called tail-anchored (TA) proteins, which are associated with different intracellular membranes, usually MOM and the endoplasmic reticulum (10). Based on the homology with Bcl-2, it has been proposed that Bax became inserted to mitochondria by its hydrophobic ␣9 helix located at its Cterminal end (for example, see Ref. 11). Several studies have shown that the Bax ␣ CT is not required for its interaction with mitochondria, whereas others have found it mandatory for Bax ␣ function (12-16). We have observed that a chimeric Bcl-XL construct in which the natural Bcl-XL CT was substituted by t...

TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax

“…12 Numerous study have shown that in a simple cell-free assay using isolated mitochondria and purified or in vitro translated (IVT) Bax could accurately reproduce early events of the Bax-induced permeabilization of the mitochondria. 10,[13][14][15] In order to elucidate the receptors involved in Bax association with mitochondria, we subjected isolated mitochondria to a mild proteolysis by trypsin using a protocol described earlier for the identification of components of the outer membrane import machinery. 16 Isolated rat liver mitochondria were treated with 0, 5 or 50 mg/ml trypsin for 30 min at 41C (see Materials and Methods) before immunoblot analyses of selected mitochondrial proteins.…”

“…Ha1 and Ha9). 9,10 With respect to the localization of these helices, the Ha1 of Bax was fused at the N-T of the RFP to form a Ha1-RFP construct while Ha9 and a Ha9 mutant containing a single deletion of S184 9 were fused to the C-T of RFP to form the RFP-Ha9 and RFP-Ha9 DS184 constructs.…”

“…Point mutations in the Ha1 of Bax or the overexpression of a chimera protein in which this helix was fused to the cytosolic Red Fluorescence Protein inhibited Baxdependent apoptosis. 10 Received 12. These two authors contributed equally to this work.…”

“…2 The mitochondrial targeting signals of Bcl-2 and Bcl-Xl have been unambiguously assigned to their C-T. 8 However, the situation seems to be more complex for Bax since, despite the homology between the C-T of Bcl-2 and Bax, the C-T of Bax, which encompasses the Ha9, does not have conventional mitochondrial targeting features as it cannot, single-handedly, support the translocation of a reporter protein to the mitochondria. 9,10 However, mutation of specific amino acids or post-translational modifications such as phosphorylation can render this C-T competent for mitochondrial addressing. 11 We have recently shown that Bax contains a functional mitochondrial-addressing signal in its N-T, which encompasses Ha1.…”

“…Point mutations in the Ha1 of Bax or the overexpression of a chimera protein in which this helix was fused to the cytosolic Red Fluorescence Protein inhibited Baxdependent apoptosis. 10 We have attempted to identify mitochondrial receptors to Bax and found that translocase of the outer membrane of mitochondria TOM22, a receptor for nuclear encoded mitochondrial proteins, was a receptor for Bax Ha1. The functional inhibition of this interaction interfered with Bax mitochondrial addressing and apoptosis underlining the importance of this interaction in cell death.…”