Abstract
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.
Highlights
As shown in Fig. lA, double immunostaining of these cells the COOH-terminal 22 amino acids of Bcl-2 abrogated with monoclonal antibodies against human Bcl-2 [4] and huprotein targeting, whereas fusion of this domain to theman MOM
COOH terminus of dihydrofolate reductase resulted in extensive co-distribution of the two antigens throughout the targeting and insertion of the hybrid protein into the cytoplasm (Fig. L4)
NH2-terminal signal anchor of the mitochondrial outer space, sulfite oxidase, was sensitive to trypsin only at signifimembrane protein in yeasMt,as7Op.A synthetic peptide cantly higher concentrations of detergent (Fig. 1B).Bcl-2 and comprising the Mas7Op signal anchor sequence effec- sulfite oxidase each contain numerous arginine anlydsine resitively 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
Summary
We show that the transmembrane domain of human Bcl-2functions as amitochondrialsignalanchor sequence that targets and inserts the protein into the RESULTS AND DISCUSSION RL.7 is a human lymphoma cell line that expresseshigh outer membrane in anNo,&& orientation, leaving the levels of Bcl-2, which is predominantly located in mitochondria bulk of the polypeptide facing the cytosol. Double immunostaining of these cells the COOH-terminal 22 amino acids of Bcl-2 abrogated with monoclonal antibodies against human Bcl-2 [4] and huprotein targeting, whereas fusion of this domain to theman MOM
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