Differences in the Association of BH3-Only Proteins to Biological Membranes

Abstract

Apoptosis, a prevalent mechanism of programmed cell death, is regulated by the Bcl-2 protein family. The balance between pro- and anti-apoptotic Bcl-2 members in the mitochondrial outer membrane (MOM) protects or triggers MOM permeabilization. Bcl-2 homology-3 (BH3)-only proteins participate in this process activating pro-apoptotic effectors and promoting permeabilization of the MOM. The membrane association of BH3-only proteins is controversial due to the lack of a canonical carboxyl-terminal (C-terminal) transmembrane (TM) domain. We used an in vitro transcription/translation system to study the insertion capacity of these hydrophobic C-terminal regions of the BH3-members Bik, Bim, Noxa, Puma, Bmf and Hrk into microsomal membranes, and an Escherichia coli complementation assay to validate our results in bacterial cells. Furthermore, we have fused these poorly hydrophobic regions to the GFP as C-terminal tails to investigate subcellular sorting in mammalian cells. Our approaches provide additional tools for testable descriptions of membrane integration, allowing further refinement for the understanding of the core mechanisms of the regulation of membrane permeabilization by Bcl-2 family members.