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

Abstract

The association of Bax with mitochondria is an essential step in the implementation of apoptosis. By using a bacterial two-hybrid assay and crosslinking strategies, we have identified TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), as a mitochondrial receptor of Bax. Peptide mapping showed that the interaction of Bax with TOM22 involved the first alpha helix of Bax and possibly two central alpha helices, which are homologous to the pore forming domains of some toxins. Antibodies directed against TOM22 or an antisense knockdown of the expression of TOM22 specifically inhibited the association of Bax with mitochondria and prevented Bax-dependent apoptosis. In yeast, a haploid strain for TOM22 exhibited a decreased expression of TOM22 and mitochondrial association of ectopically expressed human Bax. Our data provide a new perspective on the mechanism of association of Bax with mitochondria as it involves a classical import pathway. Apoptosis is a cell death program, which is central in many aspects to metazoan cell physiology and pathology. 1 Proteins of the Bcl-2 family are key players in the execution phase of apoptosis and their main functions is to control the release of apoptogenic proteins, such as cytochrome c (cyt c) and apoptosis inducing factor (AIF) from the mitochondria. 2 The Bcl-2 family is composed of antiapoptotic members such as Bcl-2 and proapoptotic proteins such as Bax or Bak, which share several domains of homology called BH. 1 In addition to these proteins, a third Bcl-2-related family of proteins have been identified as major amplifiers and/inducers of apoptosis. 2 The latter family has a homology to Bcl-2 limited to the BH3 domain and thus is called the BH3 only proteins. 2 The double knockout of Bax and Bak renders cells completely resistant to cell death 2 highlighting the essential role of these proteins during apoptosis.The determination of the solution structure of Bax showed its organization around nine a-helices (Ha1 to Ha9) 3 of which Ha2 contained most of the BH3 domain and Ha5 and Ha6, a putative pore forming domain that has been shown to be involved in the integration of Bax into the membrane. 4,5 Bax resides in an inactive state in the cytosol in many resting cells and is translocated to the mitochondria at the onset of apoptosis. 6 This translocation is associated with major conformational changes in Bax as both the amino-terminal end (N-T) and carboxy terminal end (C-T) become exposed facilitating its mitochondrial addressing and membrane insertion. 5,7 Bax mitochondrial membrane insertion and oligomerization is closely associated with the release into the cytosol of several intermembrane space proteins such as cyt c, second mitochondrial apoptotic factor (Smac) and AIF. 2 The nature of the different stimuli involved in the activation or in the inhibition of Bax is still largely unknown although this step remains one of the most critical points of control of the apoptotic program during which therapeutic interventions can be envisaged. 2 The mit...

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“…The experiments were performed as described in Bellot et al 13 Briefly, the peptide membranes were prepared on a MultiPep automated peptide synthesizer (Intavis AG, Köln, Germany). Bax sequence was divided into 12-mer peptides with a two amino acid frame shift.…”

“…13 In this assay, the whole sequence of Bax was decomposed into 12-mer peptides (overlapping by 10 residues) covalently bound to a nitrocellulose membrane, which was incubated with biotinylated PGE 2 . Figure 1c shows that biotinylated PGE 2 was able to interact with several a-helices (Ha) in Bax but showed a maximal binding to the Ha2 (BH3 domain), the loop formed between the Ha5 and Ha6 and last Ha present in its C-terminal end, Ha9.…”

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“…Most bear a C-terminal hydrophobic sequence that targets and/or anchors them to those membranes, but full integration of the multidomain proteins probably involves insertion of additional helices in the molecules (Kim et al, 2004;Annis et al, 2005), and that also might hold for tBid, which is myristoylated (Zha et al, 2000) but lacks a C-terminal anchor. Integration into the outer mitochondrial membrane may require components of the general import machinery such as TOM22 or TOM70 (Bellot et al, 2006;Chou et al, 2006).…”