Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence

The protooncogene product Bcl-2 is an integral membrane protein that functions as a suppressor of programmed cell death. It contains a single predicted transmembrane segment located at its COOH terminus. Here, we show that the transmembrane domain of human Bcl-2 functions as a mitochondrial signal anchor sequence that targets and inserts the protein into the outer membrane in an Ncyto-C(in) orientation, leaving the bulk of the polypeptide facing the cytosol. Deletion of the COOH-terminal 22 amino acids of Bcl-2 abrogated protein targeting, whereas fusion of this domain to the COOH terminus of dihydrofolate reductase resulted in targeting and insertion of the hybrid protein into the outer membrane in a manner similar to that of Bcl-2. The sequence of the hydrophobic core of the Bcl-2 signal anchor is similar to the corresponding region of the NH2-terminal signal anchor of the mitochondrial outer membrane protein in yeast, Mas70p. A synthetic peptide comprising the Mas70p signal anchor sequence effectively competed for insertion of Bcl-2 into the outer membrane but had no effect on the comparatively low association that Bcl-2 makes with endoplasmic reticulum microsomes. Insertion of Bcl-2 into the mitochondrial outer membrane is mechanistically different than its association with microsomes.