The Stability and Anti-apoptotic Function of A1 Are Controlled by Its C Terminus
Journal of Biological Chemistry
Most Bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their C-terminal portion. We found that the C terminus of the anti-apoptotic family member A1 did not function as a membrane anchor. Instead, this stretch of the protein rendered A1 highly unstable by mediating its polyubiquitination and rapid proteasomal degradation. Moreover, the domain did not only function independently of its position within the A1 protein but when transfered could even
... ed could even destabilize unrelated proteins like enhanced green fluorescent protein and caspase-3. A1 was, however, much more stable in the presence of the Bcl-2 homology-only protein BimEL, suggesting that direct interaction of A1 with pro-apoptotic members of the Bcl-2 family strongly reduces its rate of turnover. We further show that the C-terminal end of A1 also contributes to the anti-apoptotic capacity of the protein. In conclusion, our data demonstrate that the C terminus serves a dual function by controlling the stability of A1 and by amplifying the capacity of the protein to protect cells against apoptosis. EXPERIMENTAL PROCEDURES Reagents-Cycloheximide, DTT, protease inhibitor mixture (P8340), and lipopolysaccharide (Pseudomonas aeruginosa) were purchased from Sigma. Etoposide and proteasome inhibitor II were purchased from Calbiochem. Cell Culture-WEHI 231 cells (murine B cell lymphoma line), Daudi cells (human Burkitt lymphoma line), and 293T cells (human embryonic kidney cell line 293 expressing SV40 T antigen) were obtained from American Type Culture Collection. High density primary B cells (Ն1.807 g/ml) . 4 The abbreviations used are: BH, Bcl-2 homology; Casp3, caspase-3; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; DTT, dithiothreitol; FACS, fluorescence-activated cell sorter; E3, ubiquitin-protein isopeptide ligase; MCS, multiple cloning site.