(A) The sequence in Siva-1 corresponding to the predicted amphipathic helix (Top), its pinwheel representations (Middle), and Connoly space-filling models (Bottom) are shown. The hydrophobic face of the helix is shown in the model. The hydrophobic (yellow), the positively charged (red), and the negatively charged (purple) amino acids are shown. The number of the amino acid corresponds to the numbering shown in the pinwheel representation, with one corresponding to 36 and 20 to 55. (B) Intracellular distribution of expressed Siva-1-HA in HeLa cells was determined by using anti-HA-rhodamine antibody and Mitotracker Green-FM. Siva-1 is predominantly in the cytoplasm, (Upper Right, red), the green specks represent mitochondria in the same cell (Upper Left), and the superimposition of the two suggests localization of Siva-1 to mitochondria (Lower Left). The morphology of the cell is shown on the lower right. (C) Siva-1 is expressed mostly in DP murine thymocytes as evidenced from multicolor flow cytometry (Right). The cells treated under similar conditions with isotype control or secondary antibodies failed to label the cells (Left).

(A) Direct binding between BCL-X_L and Siva-1. Recombinant GST-Siva-1 and GST when mixed with soluble BCL-X_L resulted in a dose-dependent direct interaction between GST-Siva-1 and BCL-X_L, despite GST being far in excess of GST-Siva-1. (B) Recombinant purified GST-Siva-1 and GST-BCL-X_L proteins individually could be used to coprecipitate GFP-BCL-X_L and GFP-Siva-1, respectively, from cell lysates. Cos cells were transiently transfected with pEGFP-Siva-1 or pEGFP-BCL-X_L, lysed, and mixed with equivalent amounts of either GST-Siva-1 or GST-BCL-XL proteins. The GST protein complexes were precipitated as described in Methods and immunoblotted with anti-GFP and anti-BCL-XL antibodies. The first lanes (both Left and Right) represent the relative amounts of the GFP proteins expressed in the WCLs. (C) GST-Siva-1 but not GST or GST-Siva-2 binds to GFP-BCL-X_L. Cos cells were transiently transfected with GFP-BCL-X_L and GST- or GST-Siva-1- or GST-Siva-2-expressing plasmids. After 48 h, the cells were lysed, and the GST protein complexes were collected and subjected to SDS/PAGE followed by immunoblotting with anti-GFP antibody. As shown in the top left hand corner, a significant amount of GFP-BCL-X_L was seen only in the lane corresponding to GST-Siva-1. GFP itself did not bind to either GST-Siva-1 or GST-Siva-2, as shown by cotransfection of the empty GFP plasmid with GST-Siva-1 or GST-Siva-2. The same blot was stripped and probed with ant-GST antibody to demonstrate that various GST proteins are indeed precipitated (Top Right). The relative levels of expressed GFP and GST proteins in the WCLs are also shown (Bottom, Left and Right, respectively).

(A) HA-tagged Siva-1 and GFP-BCL-X_L were expressed together in HeLa cells and stained with anti-HA-rhodamine antibody. Most of the expressed Siva-1 (red) and BCL-X_L (green) colocalize to the same intracellular compartment as seen in the superimposed picture (yellow). (B) The kinetics of natural Siva-1/BCL-X_L complexes in murine thymocytes after CD3 crosslinking is shown. Thymocytes obtained from thymuses of newborn mice were cultured on plates coated with anti-mouse CD3 antibody for the indicated times. Cells (100 × 10⁶ per sample) were lysed and immunoprecipitated with either anti-rabbit Ig beads (control) or anti-BCL-X_L antibody-linked beads and subjected to immunoblotting with anti-Siva rabbit polyclonal antibody. The amount of coprecipitated Siva-1 increased by 30′ and then decreased, and in the control lane, it did not reveal the presence of significant amount of Siva-1. Immunoblotting with the secondary antibody alone did not reveal any bands in the corresponding region shown in the figure (data not shown). (C) Hut78 cells were exposed to UV and then incubated for 30 and 60 min, lysed, and subjected to immunoprecipitations. (Upper) Immunoblot probed with anti-Siva antibody. The same blot was stripped and probed with anti-BCL-X_L antibody (Lower). The vertical labels represent various immunoprecipitations. In Hut78 cells not exposed to UV radiation, a significant amount of Siva-1 was found to be associated with BCL-X_L (lane 2, Upper), and the reverse can be seen in the lower panel (lane 1), wherein BCL-X_L is coprecipitated with Siva-1 in anti-Siva immunoprecipitates. Secondary antibody-conjugated beads alone or anti-BCL-2 antibody immunoprecipitates did not bring down Siva-1 or BCL-X_L. The amount of Siva-1 in BCL-X_L immunoprecipitate increased slightly by 30 min after exposure to UV radiation, and then decreased by 60 min to undetectable levels (Upper); however, at this time point, BCL-X_L could still be found in Siva-1 immunoprecipitate (Lower). The expression of endogenous Siva-1 and BCL-XL in the WCLs is also shown (last lanes, Upper and Lower).

(A) Deletion of the SAH region in Siva-1 but not an unrelated downstream region results in loss of BCL-X_L binding. Various plasmids were coexpressed with pEGFP-BCL-X_L in Cos cells and the lysates were subjected to protein precipitations by using the glutathione beads. α-GFP immunoblot (Top) demonstrates coprecipitation of GFP-BCL-X_L with the control deletion (GST-Siva-1Δ130–149) but not SAH deletion mutant of Siva-1 (GST-Siva-1Δ36–55). Relative levels of GST-fusion proteins in the WCLs (Middle). Expression of GFP-BCL-X_L (Bottom). (B) SAH peptide but not excess of irrelevant peptide competes off GFP-BCL-X_L bound to GST-Siva-1. To the lysates containing expressed GST-Siva-1 and GFP-BCL-X_L, various concentrations of the SAH peptide or irrelevant peptide (200 μM) were added, and the GST-Siva-1 protein complexes were immunoblotted with anti-GFP antibody. The GFP-BCL-X_L bound to GST-Siva-1 (lane 1) was competed off completely at 100 μM SAH peptide (lane 2) but not with irrelevant peptide at 200 μM (lane 5). GST alone did not coprecipitate any GFP-BCL-X_L (lane 6). (C) The left-hand panel represents the relative expression of GFP-BCL-X_L; the right-hand panel represents the relative expression of the GST and GST-Siva-1 proteins in the WCLs used in the competition experiment shown in B.

(A) GFP but not GFP-BCL-X_L stable MCF7 transfectants are highly susceptible to UV radiation-induced apoptosis. (B) Transient expression of GST-Siva-1 but not GST or GST-Siva-2 results in loss of protection against UV radiation-induced apoptosis in BCL-X_L transfectants. (C) Representative pictures of apoptotic nuclei seen in B by staining with Hoechst are shown. (D) Deletion of the SAH domain in Siva-1 (Δ36–55) but not an irrelevant region (Δ130–149) results in the complete loss of its ability to suppress BCL-X_L function.