Mitochondria-mediated apoptosis is regulated by proteins of the Bcl-2 superfamily, most of which contain a C-terminal hydrophobic domain that plays a role in membrane targeting. Experiments with BNIP3 have implicated the transmembrane (TM) domain in its proapoptotic function, homodimerization, and interactions with Bcl-2 and Bcl-x L . We show that the BNIP3 TM domain self-associates strongly in Escherichia coli cell membranes and causes reversible dimerization of a soluble protein in the detergent SDS when expressed as an in-frame fusion. Limited mutational analysis identifies specific residues that are critical for BNIP3 TM selfassociation in membranes, and these residues are also important for dimerization in SDS micelles, suggesting that the self-association observed in membranes is preserved in detergent. The effects of sequence changes at positions Ala 176 and Gly 180 suggest that the BNIP3 TM domain associates using a variant of the GXXXG motif previously shown to be important in the dimerization of glycophorin A. The importance of residue His 173 in BNIP3 TM domain dimerization indicates that polar residues, which have been implicated in self-association of model TM peptides, can act in concert with the AXXXG motif to stabilize TM domain interactions. Our results demonstrate that the hydrophobic C-terminal TM domain of the pro-apoptotic BNIP3 protein dimerizes tightly in lipidic environments, and that this association has a strong sequence dependence but is independent of the identity of flanking regions. Thus, the transmembrane domain represents another region of the Bcl-2 superfamily of proteins that is capable of mediating strong and specific protein-protein interactions.The Bcl-2 superfamily of proteins plays a central role in regulating mitochondria-mediated apoptosis; the Bax subfamily promotes apoptosis, whereas the Bcl-2 subfamily protects against apoptosis (reviewed in Ref. 1). Protein-protein interactions between Bax and Bcl-2 subfamily members help determine cell fate (2, 3) and have accordingly been the subject of intensive study. Four regions of sequence homology, the BH1 through BH4 domains, contribute to the structure and function of these proteins, and the BH3 domain is particularly implicated in heterodimerization events (4 -9). Peptide and small molecule inhibitors of these protein-protein interactions can modulate apoptosis, further demonstrating the functional and pharmaceutical importance of these contacts (10 -13).Differentiation and development in metazoans requires celland signal-specific inputs to a functional Bcl-2/Bax checkpoint. The pro-apoptotic "BH3-only" proteins, which show homology to the Bcl-2 superfamily through the BH3 domain alone (14), are expressed or activated in specific tissues and in response to certain stimuli, making them candidates for connecting diverse signaling pathways to the ubiquitous apoptosis effector machinery (15, 16). BNIP3 (Bcl-2/19-kDa interacting protein 3) is a BH3-only protein whose expression and pro-apoptotic activity is induced following hyp...

SummaryWe present an implementation of the TOXCAT membrane protein self-association assay that measures the change in apparent free energy of transmembrane helix dimerization caused by point mutations. Quantifying the reporter gene expression from cells carrying wild type and mutant constructs shows that single point mutations that disrupt dimerization of the transmembrane domain of glycophorin A reproducibly lower the TOXCAT signal more than one hundred-fold. Replicate cultures can show up to three-fold changes in the level of expression of the membrane bound fusion construct, and correcting for these variations improves the precision of the calculated apparent free energy change. The remarkably good agreement between our TOXCAT apparent free energy scale and free energy differences from sedimentation equilibrium studies for point mutants of the glycophorin A transmembrane domain dimer indicate that sequence changes usually affect membrane helix-helix interactions quite similarly in these two very different environments. However, the effects of point mutations at threonine 87 suggest that intermonomer polar contacts by this side chain contribute significantly to dimer stability in membranes but not in detergents. Our findings demonstrate that a comparison of quantitative measurements of helix-helix interactions in biological membranes and genuine thermodynamic data from biophysical measurements on purified proteins can elucidate how changes in the lipidic environment modulate membrane protein stability.