A Novel Inhibitory Mechanism of Mitochondrion-Dependent Apoptosis by a Herpesviral Protein
Figure 7
vMAP Interaction with VDAC1 Inhibits Cytochrome c Release
(A) vMAP contains two VDAC1-interacting regions (dark grey boxes). The left panel shows the schematic diagram of vMAP interaction with VDAC1. (Right panel) GST fusions containing various vMAP sequences were expressed and purified from E. coli. 293T cells were lysed in CHAPS buffer and subjected to in vitro GST pull-down, followed by immunoblotting with anti-VDAC antibody. The bottom panel shows the Coomassie blue staining of GST fusion proteins. Lanes 1–4 correspond to the GST fusions shown by the left diagram.
(B) The LLxL repeat sequences of vMAP are required for VDAC1 interaction. (Top box) The boxed sequences are the two LLxL repeats of vMAP. GST or GST fusion proteins were used to bind to VDAC1 from 293T cell lysates in CHAPS buffer as described in (A). Protein precipitates were analyzed by immunoblotting with anti-VDAC antibody (top blot) and Coomassie blue staining of GST fusion proteins (bottom blot).
(C) vMAP interacts with VDAC1 in stable cell lines. NIH3T3 cells stably expressing vector, vMAP, or its mutants were used for immunoprecipitation with anti-vMAP serum or pre-immune (Pre) serum, followed by immunoblotting with anti-VDAC antibody (top panel) or anti-vMAP serum (middle panel). WCLs were analyzed by immunoblotting with anti-vMAP serum and anti-VDAC antibody (bottom two panels).
(D) vMAP interacts with VDAC1 in γHV-68-infected cells. WCLs of mock (M)- or γHV-68 (γ)-infected (MOI = 1) cells were precipitated with anti-vMAP or preimmune serum, followed by immunoblotting with anti-VDAC antibody (top panel) or anti-vMAP serum (middle panel). WCLs were analyzed by immunoblotting with anti-vMAP serum and anti-VDAC antibody (bottom two panels).
(E) vMAP inhibits cytochrome c release upon ST treatment. NIH3T3/puro, NIH3T3/vMAP, or NIH3T3/vMAP L/A cells were treated with ST (1 μM) for 4 h and WCLs were sequentially centrifuged to obtain cytosolic (Cyto) and mitochondrion-enriched heavy membrane (HM) fractions. Polypeptides (20 μg) from each fraction were analyzed by immunoblotting with antibodies to cytochrome c (Cyt C), COX4, and actin.