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First published online 31 July 2007
doi: 10.1242/jcs.006197

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The tail-anchoring domain of Bfl1 and HCCS1 targets mitochondrial membrane permeability to induce apoptosis

¹ Department of Physiology and Biophysics, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA
² Department of Pathology, Tumor Immunity Medical Research Center and Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea

View larger version (45K): [in this window] [in a new window]   Fig. 1. The amphipathic tail-anchoring peptide (ATAP) is conserved in Bfl1 and HCCS1. (A) Schematic genomic structures of BCL2A1 (BFL-1) and HCCS1 genes on human chromosome 15q24.3 and 15q25.1, respectively. Black bars indicate exons and red bars indicate conserved exon-3 of BCL2A1 and HCCS1 genes. (B) Alignment of exon-3 sequences from BCL2A1 and HCCS1 genes. Identical sequences are shaded gray and single base gap is shaded black. Stop codons are yellow. (C) Primary sequence comparison of the TA region of Bfl1 and HCCS1. Horizontal red bar indicates the ATAP sequence of Bfl1 and HCCS1. Green lines indicate predicted -helical regions. Mitochondrial targeting signal (MTS) of HCCS1 is indicated by a blue line. (D) Helix-wheel diagrams of ATAP sequences of Bfl1 and HCCS1. (E) Amino acid sequence alignment of TA regions from human anti-apoptotic Bcl2 family proteins and A1, a mouse homologue of human Bfl1, and ATAP sequences.

View larger version (34K): [in this window] [in a new window]   Fig. 2. ATAP-induced apoptosis is independent of Bax and Bak activities. (A) Transient expression of Flag-ATAP induced caspase-dependent cell death of HEK293 cells. Cells were co-transfected with 1 µg of mock or Flag-ATAP expression plasmid with 0.1 µg pCMV--gal in the absence or presence of 50 µM OPH. Cell viability was measured by -galactosidase activity relative to control cells transfected with mock plasmid and the pCMV--gal reporter plasmid (left). The relative expression levels of the Flag-ATAP peptide was determined by western blotting with an antibody against the Flag tag epitope (right). (B,C) Transient expression of GFP-ATAP-induced apoptotic nucleus morphology and sub-G1 population in a caspase-dependent manner. GFP-ATAP-transfected HeLa cells were fixed, stained with DAPI and observed under fluorescence microscope 24 hours after transfection (B). HEK293 cells were transiently transfected with the indicated expression plasmids. 18 hours after transfection, cells were harvested, fixed, and stained with PI. The DNA content of GFP-positive cells was then analyzed by flow cytometry (C). (D) Transient expression of GFP-ATAP induced acute cell death in a variety of cancer cells, including HEK293, HeLa, caspase-3 deficient MCF-7 cells and BMK D3 cells derived from the kidney of neonatal knockout mice for Bax and Bak genes. 24 hours after transfection, cell survival was measured by PI exclusion. Representative images taken from HEK293 cells are shown. (E) The percentage of surviving cells was determined by the ratio of PI-negative cells to total GFP-positive cells. About 300 cells from three different fields were scored. Data are expressed as the mean ± s.e. Bars, 10 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 3. The pro-apoptotic activity of ATAP requires an amphipathic property. (A) Schematic representation of the GFP-ATAP constructs in which point mutations were introduced into the hydrophobic rich (HR) region of the ATAP. (B) Cell survival was measured by PI exclusion in the HEK293 cells transfected with 1 µg GFP-ATAP mutant constructs 24 hours after transfection. (C) Flag-mHR3 peptide showed reduced pro-apoptotic activity. HEK293 cells were co-transfected with 0.1 µg pCMV--gal reporter plasmid and 1 µg of the Flag-ATAP or Flag-mHR3 expression plasmids. 24 hours after co-transfection, cell viability was measured by -galactosidase activity (left). The relative expression levels of the Flag-tagged peptides were determined by western blotting with an antibody against the Flag tag epitope (right). (D) Involvement of amphipathic nature of ATAP in the apoptotic function of HCCS1. HEK293 cells were co-transfected with 0.1 µg pCMV--gal reporter plasmid and 1.0 µg HCCS1-GFP or HCCS1-GFP (E46Q/E53Q) containing mutations corresponding to mHR3 of the Bfl1 ATAP. 24 hours after co-transfection, cell viability was measured by -galactosidase activity. Data are expressed as the mean ± s.e.

View larger version (59K): [in this window] [in a new window]   Fig. 4. The pro-apoptotic activity of ATAP involves targeting to mitochondria. (A) Schematic representation of the GFP-ATAP constructs in which point mutations were introduced into the flanking regions of the ATAP. (B) Cell survival was measured by PI exclusion in the HEK293 cells transfected with 1 µg GFP-ATAP mutant constructs 24 hours after transfection. (C) Subcellular localization of ATAP mutants fused with GFP. HeLa cells were transfected with 0.5 µg of the indicated plasmids. Cell culture was performed in the presence of 50 µM OPH to prevent rapid cell death. 18 hours after transfection, cells were incubated in medium containing 50 nM MitoTracker for 2 hours and fixed using 4% paraformaldehyde. Localization of GFP fusion proteins was observed using confocal microscopy. Bar, 10 µm. (D) Cellular effects of GFP-ATAP mutants on apoptosis. HEK293 cells were transiently transfected with the indicated plasmids expressing ATAP mutants fused with GFP. 18 hours after transfection, cells were harvested, fixed, and stained with PI. The DNA content of GFP-positive cells was then analyzed by flow cytometry (upper panel). DNA fragmentation was also analyzed by electrophoresis on 2% agarose gels (lower panel). SM, 100 bp ladder size marker. (E) Time-course effect of GFP-ATAP and GFP-ATAP mutants on HEK293 cells. Cell survival was measured by PI exclusion in the HEK293 cells transfected with 1 µg GFP-ATAP mutant constructs.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Both MTS and ATAP are involved in mitochondrial-targeting apoptosis induced by HCCS1. (A) Schematic representation of the GFP fusion proteins of HCCS1 and its deletion mutants. (B) Subcellular localization of GFP fusion proteins. HeLa cells were transfected with 0.5 µg of the indicated plasmids. Cell culture was performed in the presence of 50 µM OPH. 18 hours after transfection, cells were incubated in medium containing 50 nM MitoTracker for 2 hours and fixed using 4% paraformaldehyde. Localization of GFP fusion proteins was observed using confocal microscopy. Bar, 10 µm. (C) Cell survival was measured by PI exclusion in the HEK293 cells transfected with 1 µg GFP fusion constructs 24 hours after transfection.

View larger version (35K): [in this window] [in a new window]   Fig. 6. ATAP induces the loss of mitochondrial membrane potential and perturbs membrane permeability in planar lipid bilayers. (A) Effect of GFP-ATAP on the mitochondrial membrane potential. Mitochondrial outer membrane potential was observed using MitoTracker Red (CM-H2 TMRos) dye. HeLa cells were transfected with GFP-ATAP, GFP-mHR7 or GFP-TAxL and cultured in the presence of 50 µM OPH. 24 hours after transfection, cells were incubated in medium containing 50 nM of MitoTracker for 2 hours and observed under a fluorescence microscope. Bar, 10 µm. (B) Green (GFP) and red (MitoTracker) double-positive cells were quantified from about 300 GFP-positive cells from three different fields. Data are expressed as the mean ± s.e. (C) Effect of ATAP on cytochrome c release. Left panel, HEK293 cells were transfected with 1 µg of pEGFP (lane 1), pEGFP-ATAP (lane 2) or pEGFP-mHR7 (lane 3). 20 µg mitochondria-free cytosol proteins were analyzed by western blotting using anti-cytochrome c antibody. Right panel, mitochondria membrane isolated from BMK-D3 cells were incubated with synthetic ATAP and mHR7 peptides (100 µM). ATAP induced cytochrome c release into the supernatant (S), whereas cytochrome c remained in the pellet (P) in preparations treated with mHR7 and DMSO (as a control). (D) Effect of synthetic ATAP and mHR7 peptides on the membrane permeability of planar lipid bilayer. The peptides (11.1 µM) were added to the cis chamber. Current traces at the corresponding holding potentials were measured from recording solution of 200 mM KCl (cis) and 50 mM NaCl (trans). Data are representative of n=5 for ATAP and n>35 for mHR7.

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