Apoptosis triggered by a deletion mutant that lacks the first 29 amino acids (Bax 30–192) was poorly inhibited by Bcl-x_L (Figure 1A) or Bcl-2 (Figure 1B) in the Jurkat T-cell line. Since Bcl-2 or Bcl-x_L function may be modulated by other pro-apoptotic proteins present in mammalian cells, we turned to the heterologous system of Drosophila melanogaster, which expresses only one pro- and one anti-apoptotic protein of the Bcl-2 family 34. We hypothesized that by expressing these proteins in Drosophila, modulation by an endogenous modifier (if indeed it occurs) will be independent of the construct being tested and will be equivalent in all conditions.

Bax-GFP, Bax 30–192-GFP and Bcl-2 were cloned into the pUAST vector and expression of the tagged proteins at the appropriate molecular weights confirmed in the Drosophila S2 cell line (Figure 2A). Transgenic fly lines of Bax, Bax 30–192 and Bcl-2 were generated by independent P-element transformation of the UAS-Bax-GFP (BA64III and BA13I), UAS-Bax30–192-GFP (DBA8II and DBA8IA) and UAS-Bcl-2 (BC57I and BC24III) constructs. Using the UAS-GAL4 system both Bax and Bax 30–192 were lethal to flies when driven in various tissues while Bcl-2 expression alone was not lethal (Table 1). This allowed us to test for the rescue of lethality by Bcl-2.

For these experiments, vg-GAL4 and twist-GAL4 lines were used to drive expression largely in the dorsal margins of wings and mesoderm respectively. When driven along with Bax (BA64III, BC57I) using vg-GAL4 (Figure 2Bleft panel) or twist-GAL4 (Figure 2B, right panel) Bcl-2 rescued Bax induced lethality whereas Bax 30–192 (DBA8II, BC57I) induced lethality was only marginally rescued. Flies expressing Bax or Bax 30–192 (using vg-GAL4) present deformity in wings to various degrees (Figure 2I). All the flies that emerged for Bax (BA64III) and Bax 30–192 (DBA8II) presented a strong wing defect (Figure 2D and 2E). Bcl-2 co expression with Bax in the double transgenic fly line BA64III, BC57I resulted in complete rescue of the wing defect (Figure 2G) while there was no change in the severe wing defect in Bax 30–192 and Bcl-2 (DBA8II, BC57I) expressing flies (Figure 2H). Additional transgenic fly lines of UAS-Bax (BA13I), UAS-Bax 30–192(DBA8IA) and UAS-Bcl-2 (BC24III) were tested for viability and wing defect using vg-GAL4 with similar results (Table 2). Thus, the experiments using the heterologous model system of Drosophila melanogaster recapitulated the observation of inefficient inhibition of Bax 30–192 by anti-apoptotic proteins in mammalian cells.

To assess the contributions of the TM domains in Bax 30–192 cytotoxicity, additional regions were deleted (Figure 3A). Apoptosis triggered by a mutant devoid of the C-terminal TM2 domain (Bax 30–171) is Bcl-2 independent, whereas apoptosis triggered by Bax 1–171 is inhibited (Figure 3B). However, it should be noted that apoptosis triggered by this construct was considerably lower than that triggered by full-length Bax thereby confounding interpretation of the data. Bax 30–105 which lacks any putative or experimentally proven TM domain but retains the BH3 domain triggered high levels of apoptotic damage, which was blocked by Bcl-x_L or Bcl-2 (Figure 3C).

We then tested the deletion mutants for regulation by Bcl-x_L in the HEK cell line as the larger size and adherent nature of the cells facilitates assessment of apoptotic damage and subcellular distribution of GFP-tagged proteins. Bax induced apoptosis was effectively blocked by Bcl-x_L; apoptosis triggered by Bax 30–192 was poorly inhibited, and apoptosis triggered by Bax 30–105 was blocked by ectopically expressed Bcl-x_L in this cell line (Figure 3D). Jurkat cells express lower levels of Bax protein compared to the HEK cell line arguing against interactions with endogenous Bax accounting for the responses of the deletion mutants.

The first alpha helix (amino acids 16–35) in the Bax N-terminus is implicated in interactions with activator BH3-only proteins 152829. In apoptotic cells, Bax clusters co-localized with Bid or Bim (Figure 4A and 4B, upper panel). In cells expressing Bcl-x_L, Bid and Bim are evenly distributed and do not overlap Bax-GFP (Figure 4A and 4B, lower panel). Bax 30–192-GFP tends to coalesce into a large cluster and overlaps with regions of intense staining with Bid or Bim (Figure 4C and 4D upper panel). However, there is no change in the largely punctate distribution of Bax 30–192 or the overlap with Bid or Bim in cells co-expressing Bcl-x_L (Figure 4C and 4D, lower panel).

Since Bax 30–105 triggered Bcl-x_L dependent apoptosis although it did not localize to mitochondria (Figure 5D), we asked if it triggered the activation of endogenous Bax. Changes in Bax conformation consistent with its activation reveal an epitope in the N-terminus that can be detected using a specific antibody clone 6A7 35. Bax 30–105 lacks the relevant section of the N-terminus, and 6A7 reactivity in cells expressing Bax 30–105 indicated the activation of endogenous Bax (Figure 4E). This reactivity was lost in cells that express Bax 30–105 and Bcl-x_L (Figure 4F). Further, endogenous Bax as detected by the reactivity of clone 6A7 colocalized with Bid and Bim and this was regulated by Bcl-x_L (Figure 4E and 4F).

A construct that included the TM1 domain comprising the α5–α6 helices, but not the N-terminal 29 amino acids (Bax 30–146) (Figure 5A) triggered Bcl-x_L independent cytotoxicity (Figure 5B) indicating that the TM1 domain interfered with the anti-apoptotic function of Bcl-x_L. Since Bax function converges on the activation of caspase-9 36 we analyzed apoptotic damage induced by Bax, Bax 30–146 or Bax 30–105 when co-expressed with a construct that is a dominant-negative inhibitor of caspase-9 (DNC9) 3738. Apoptosis triggered by all the mutants tested, including Bax 30–192 (data not shown) was blocked by the co-transfection of DNC9 in HEK cells (Figure 5C) and in the Jurkat cell line (data not shown). The data show that all the constructs tested activate a caspase-9 mediated apoptotic cascade as has been established for Bax.

Sub-cellular fractionation of HEK cells (Figure 5D) transfected with the relevant constructs showed that Bax is present in both the cytosolic and mitochondrial fractions, whereas increased amounts of Bax 30–146 was detected in the mitochondrial fractions (Figure 5D, upper and middle panel). In many experiments, the distribution of Bax 30–146 and Bax 30–192 was skewed to the mitochondrial fraction (not shown). Expectedly, Bax 30–105 was consistently detected only in the cytosolic fraction. The mitochondrial matrix protein Cox-4 was used to ascertain purity of the fractions.

Subsequently we assessed the cellular distribution of the constructs using GFP to report on Bcl-x_L regulation of Bax localization. In the absence of exogenous Bcl-x_L, the distribution of Bax, Bax 30–146 and Bax 30–192 is punctate in apoptotic cells whereas, Bax 30–105 is diffuse (Figure 6A). In cells co-transfected with Bcl-x_L (RFP-Bcl-x_L), Bax distribution is rendered diffuse (Figure 6B) as opposed to Bax 30–146 or Bax 30–192, where the punctate distribution is largely unchanged by Bcl-x_L (Figure 6B). The distribution of Bax 30–105 was not changed by Bcl-x_L (Figure 6B). The distribution of Bax in punctate or diffuse patterns in the presence or absence of Bcl-x_L is plotted in Figure 6C. The values are derived from an assessment of approximately 200 cells in random fields in each experiment. This experiment shows that in Bax constructs which lack the first 29 amino acids Bcl-x_L regulation of cellular distribution is compromised.

Ectopically expressed Bcl-x_L immunoprecipitated cotransfected Bax, Bax 30–146 or Bax 30–105 (Figure 6D) indicating that association with Bcl-x_Lis not impaired in the absence of the first twenty nine residues in Bax protein.

Since Bcl-x_L poorly regulated Bax 30–146 or 30–192 despite the association with the protein, we tested if the N-terminus can modulate this function. Apoptosis triggered by Bax 1–146 which includes the N-terminus and the TM1 domain was efficiently blocked by RFP-Bcl-x_L (Figure 7A). Bax 1–146 immunoprecipitates Bcl-x_L and is detected in both mitochondrial and cytosolic fractions (Figure 7B). Furthermore, the punctate distribution of Bax 1–146 in apoptotic cells redistributes to a diffuse pattern in cells that co-express Bcl-x_L (Figure 7C). The distribution of Bax 1–146 overlaps significantly with that of Bcl-x_L (Figure 7C). The change in distribution of Bax 1–146 in response to co-expressed Bcl-x_L is shown (Figure 7D). In a construct that lacks the TM domains, the N-terminus did not modulate Bax-BH3 function (Figure 7E).

Phosphorylation dependent modifications that change protein conformation regulate the apoptotic function of Bcl-2 family proteins. Bax-induced apoptosis is inhibited by the serine-threonine kinase Akt (Figure 7F and 7H). This regulation was compromised in a Bax construct (Bax-Ala3) with alanine substitutions of the Ser/Thr residues (T14, S15, S16) in the N-terminus which was refractory to inhibition by Akt (Figure 7F and 7H). The modification in the N-terminus however, did not interfere with Bcl-x_L inhibition of Bax-Ala3 induced apoptosis (Figure 7G and 7I).

The deletion mutants were tested in a cellular system where Bax activity is regulated by externally applied stimuli 1139. In Cos-7 cells, staurosporine (STS) triggered the translocation of ectopically expressed Bax and was a potent inducer of Bax-mediated cytotoxicity, which was inhibited in cells co-expressing Bcl-x_L (Figure 8A). Bax 30–192 and Bax 30–146 localized into puncta (data not shown) or large clusters and triggered high levels of apoptotic damage which were not further enhanced by STS (Figure 8B and 8C) or inhibited by Bcl-x_L (Figure 8B and 8C). Bax 1–146 is largely diffuse but also localizes as puncta and is a potent inducer of Bcl-x_L dependent cytotoxicity in response to STS (Figure 8D). Bax 30–105 triggered apoptosis in the absence of STS although Bcl-x_L inhibited apoptotic damage in the presence or absence of the apoptotic stimulus (Figure 8E).

The data thus far suggested that apoptosis triggered by Bax 30–146 and Bax 30–192 should not depend on endogenous Bax or Bak, whereas Bax 30–105 induced apoptosis may depend on these proteins. MEF deficient for both Bax and Bak were therefore tested for susceptibility to apoptosis by the Bax deletion mutant constructs. Confirming these predictions Bax and Bax 30–192 but not Bax 30–105 triggered apoptosis in Bax/Bak deficient cells (Figure 8F).

In an attempt to elucidate structural correlates if any, of the phenotypes presented by the mutants we turned to a sequence-based analysis of the interacting proteins. The three-dimensional structures of full-length Bax sequence and deletion mutants have been obtained from homology modeling, using MODELLER (version 8v1) starting from the human protein that has 92% sequence identity. The homology model of Bax was examined using the program DIAL 40. The deletion experiments, using mutants lacking the first 29 residues, suggest that major structural alterations in full-length Bax involving the N-terminal 29 residues, influence the penultimate helix connecting TM1 and TM2 regions in the three-dimensional model of full-length Bax (Figure 9A). In order to test if there is structural equivalence between the anti-apoptotic protein Bcl-x_L and regions in Bax, we examined full-length Bcl-x_L and Bax for sub-optimal sequence similarity. A high gap penalty of 400 was employed to align the two sets of sequences using MALIGN 41. In particular, we notice that the first helix of TM1 (TM1.1 helix) and the second helix of TM1 (TM1.2 helix) have significant sequence similarity with α-5 and α-6 of Bcl-x_L and related anti-apoptotic proteins, respectively (Figure 9B). This is supported by previous structural studies where Bcl-x_L was co-crystallized with BH3-region of Bim and both helices α-5 and α-6 of Bcl-x_L in this complex interact with the BH3 of Bim 9. This suggests that the presence of TM1 region could act as structural antagonists for the interaction of Bcl-x_L with Bax. Furthermore, the BH2 region of Bcl-x_L shows local sequence similarity with the penultimate BH2 helix in Bax (Figure 9C).

Jurkat, HEK and COS-7 cell lines and the Bax-Bak deficient MEF were maintained in culture in medium supplemented with antibiotics, glutamine (Invitrogen-Gibco, Carlsbad, CA) and 5 or 10% fetal calf serum (Hyclone, Logan, UT). Antibodies were procured from the following sources: to GFP and Bim from BD Biosciences (Franklin Lakes, NJ); to Bax and Bcl-2 from Santa Cruz (Santa Cruz, CA); to Cox-4 and and Alexa Fluor 555/Cy5 conjugated secondary antibodies from Invitrogen-Molecular Probes; to Bid from Cell Signaling Technology; to Bcl-x_L from Abcam (Cambridge, UK); actin and clone 6A7 from Neomarker (Fremont, CA) respectively. Protein G agarose beads were from Pierce Biotechnology (Rockford, IL). Mouse or Rabbit True-Blot HRP (Ebiosciences, San Diego, CA) was used in the western blot analysis of immunoprecipitated proteins. All other chemicals were obtained from Calbiochem (San Diego, CA) or Invitrogen.

A mammalian expression vector encoding murine full-length Bax was the kind gift of Peter Vandenabeele (University of Gent, Belgium). Bax-GFP and the deletion mutants were generated by PCR amplification and subcloning into the pEGFP-N1 vector (BD Clontech, Mountain View, CA) with GFP in-frame at the C- terminus. The following primers were used to generate the Bax and Bax mutants: Bax/Bax 1–171/Bax 1–146 forward 5' CCG CTC GAG ATG GAC GGG TCC GGG 3' Bax 30–192/Bax 30–171/Bax 30–146/Bax 30–105 forward 5' CCG CTC GAG ATG TTC ATC CAG GAT 3' Bax/Bax 30–192 reverse 5' CCC AAG CTT GCC CAT CTT CTT CCA 3' Bax 30–171/Bax 1–171 reverse 5' CCC AAG CTT CTG CCA TGT GGG 3' Bax 30–146/Bax 1–146 reverse 5'CCC AAG CTT CTC ACG GAG GAA 3' Bax 30–105 reverse 5' CCC AAG CTT GAA GTT GCC ATC 3'. The murine Bcl-2 plasmid was obtained from Upstate (Charlottesville, VA). The dominant negative caspase 9 construct was the kind gift of C. Vincenz (University of Michigan Medical School, Ann Arbor, MI) and the human Bcl-x_L plasmid was from Richard J. Youle (National Institutes of Health, Bethesda, MD). CA-Akt was from Upstate (Charlottesville, VA). Monomeric RFP (mRFP) plasmid was originally from Roger Y. Tsien (University of California, San Diego, CA) and obtained through Satyajit Mayor (National Centre for Biological Sciences, Bangalore, India). Bcl-x_L was subcloned into the pUSEAmp mammalian expression vector and was also subcloned at the 3' end of mRFP under the CMV promoter. Mito-DsRed was obtained from V. Sriram (National Centre for Biological Sciences, Bangalore, India). The sequence of all constructs was verified by automated sequencing (Microsynth, Balgach, Switzerland). The alanine substitution mutant Bax-Ala3 (substituted on T14, S15 and S16) was generated at Everogen (Moscow, Russia).

Jurkat and 2B4 cells were transfected by electroporation. Briefly 3–5 million cells were electroporated at 250 V and 960 μF and cultured for 12 hours prior to analysis. HEK and COS-7 cells were plated at 0.4–0.5 million cells/ml and MEF at 0.3 million/ml 12–15 hours prior to transfection. Cells were transfected using lipofectaime 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Cos-7 cells were transfected with 0.2 μg of Bax constructs and 2 μg Bcl-x_L. 12–15 hours later staurosporine was added at a final concentration of 1 μM and the cultures continued for an additional 3–4 hours before analysis.

Transfected cells were harvested and stained for 3 minutes at ambient temperatures in the dark using the Hoechst 33342 (1 μg/ml) and assessed for apoptotic nuclear damage. GFP positive cells were viewed under the UV filter of the fluorescence microscope and nuclear morphology scored for a minimum of 200 cells in each experimental condition. In a minimum of two experiments in each set of data samples were scored double-blind. Statistical significance of the differences between groups was assessed by the Student's t-test and is indicated by an * in the figures.

The protocol followed has been previously described 52. Briefly, 1 million cells were incubated in 100 μl homogenization buffer (250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, pH 7.2 supplemented with 2 μg/ml aprotinin, leupeptin and pepstatin) for 15 min on ice. Cells were mechanically disrupted by passing through a 26-gauge needle 15 times followed by a 30-gauge needle 15 times. Cell lysates were centrifuged at 750 g for 10 min at 4°C to remove unlysed cells and nuclei. The supernatant was centrifuged at 12,000 g for 20 min at 4°C to obtain the pellet enriched in mitochondria and the supernatant enriched for cytosolic proteins. Equivalent volumes of cytosolic and mitochondrial fractions were used for western blot analysis to assess the relative distribution of Bax and the mutants. The densitometry analysis of western blots was performed using Image Gauge software Version-3 (Fujifilm Science, Japan)

In each group two million cells were harvested and lysed in 500 μl of CHAPS lysis buffer (1% CHAPS in 50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, pH7.4 supplemented with 2 μg/ml aprotinin, leupeptin, pepstatin, 1 mM PMSF, 1 mM NaF and 1 mM Na₃OV₄) by 3 freeze thaw cycles in liquid nitrogen and 37°C followed by an incubation (30 minutes) at 4°C on a cell rotator. The lysate was centrifuged at 3000 rpm to remove debris and unlysed cells. The supernatant was incubated with 10 μg of antibody to Bcl-x_Lat 4°C for 1 hour on a cell mixer. 80 μl of washed Protein-G agarose beads were then added to the sample and mixed by gentle rotation for another 2 hours at 4°C. Beads were pelleted by centrifugation at 1700 rpm and subsequently washed 4 times with chilled PBS. After the final wash the beads-complex was pelleted by centrifugation at 2500 rpm and boiled in SDS lysis buffer for 10 minutes before western blot analysis.

Cells washed with PBS and fixed in 4% paraformaldehyde for 15 minutes at ambient temperature were permeabilized with 0.2% CHAPS in PBS (permeabilization buffer) for 30 minutes on ice and blocked for 1 hour on ice with 5% BSA diluted in permeabilization buffer. Cells were incubated with primary antibodies diluted in blocking buffer for 1–2 hours on ice followed by a wash with permeabilization buffer. The cells were finally incubated with Alexa Fluor 555 and/or Cy5 conjugated secondary antibodies diluted in blocking buffer for 1 hour on ice. Cells were also counterstained with Hoechst 33342 before the final wash and imaged.

Cells were imaged using a Bio-Rad MRC 1024 laser scanning confocal microscope equipped with Argon-Krypton laser and 60X, NA 1.4 objective lens. Fluorescence images of cells were recorded with sequential excitation and emission conditions using appropriate optics. The acquired images were processed for co-localization analysis using Image J1.34s software. For the analysis of endogenous Bid, Bim or Bax, samples were imaged using a LSM 510 Meta laser scanning confocal microscope (Carl Zeiss, Jena, Germany) equipped with Argon, Helium-Neon lasers and 63X, NA 1.4 objective. The fluorescence images for GFP tagged, Alexa Fluor 555 and Cy5 labeled proteins were acquired with sequential excitation (488 nm, 543 nm and 633 nm respectively) and emission conditions using appropriate optics. The nuclear morphology images were acquired using two photon excitation of single plane with 750 nm pulsed laser (Spectra-Physics, CA). Images were processed for co-localization analysis using Image J1.34s and LSM Meta (Carl Zeiss, Jena, Germany) software.

Bax-GFP, Bax 30–192-GFP and Bcl-2 were cloned in the UAS vector pUAST. The resulting plasmids were injected into yw; +; Ki P(Δ2-3ry+) embryos using standard procedures. Several independent transformant lines were obtained by P-element transformation for each transgene. A minimum of two lines in each case were characterized for further analysis. Bax-GFP insertion was mapped to chromosomes III and I for BA64III and BA13I transgenic lines respectively. Bax 30–192-GFP insertion was mapped to chromosome II and I for DBA8II and DBA8IA fly lines respectively. Bcl-2 insertion was mapped to chromosome I and III for BC57I and BC24III fly lines respectively. A single copy transformant fly line for each transgene was crossed to flies carrying vestigial (vg)-GAL4, twist-GAL4, eyeless-GAL4, scalloped-GAL4 or collagen-GAL4. Flies were raised on standard Drosophila medium at 25°C.