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Mitochondrial targeting of the proapoptotic protein Bax and other tail‐anchored proteins

Action of the proapoptotic protein Bax requires a signal‐dependent targeting to the mitochondrial outer membrane. The mechanism accomplishing this targeting is unknown. A hydrophobic stretch of amino acids within the C‐terminal tail of Bax serves as the targeting signal as well as the membrane anchor. This places Bax in a class of proteins referred to as tail‐anchored proteins. We have utilized artificial tail‐anchor sequences to define features that specifically direct targeting to the mitochondrial outer membrane. An artificial sequence consisting of 16 leucines fused to a reporter protein (Green Fluorescent Protein) was used as a template for mutagenesis. It was demonstrated that increasing the polarity of this sequence, as well as introducing positive charge at the very C‐terminus, directs targeting from the ER to the mitochondria. Circular dichroism studies were also performed on these sequences. These demonstrate that mitochondrial‐targeted tail‐anchor has a greater propensity to form á‐helices in solution compared to ER‐targeted tail‐anchor. Currently this data is being used as a basis to examine the structural requirements of the Bax mitochondrial targeting signal. Site‐directed mutagenesis is being employed to determine specific amino acid requirements within the tail. The importance of these elements is being assessed by measuring targeting efficiency and apoptotic activation capability of Bax. Nuclear Magnetic Resonance (NMR) is also being used to assess the 3‐dimensional structure of the Bax tail‐anchor.