The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling - PubMed (original) (raw)

The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling

M Mancini et al. J Cell Biol. 1998.

Abstract

Caspase-3-mediated proteolysis is a critical element of the apoptotic process. Recent studies have demonstrated a central role for mitochondrial proteins (e.g., Bcl-2 and cytochrome c) in the activation of caspase-3, by a process that involves interaction of several protein molecules. Using antibodies that specifically recognize the precursor form of caspase-3, we demonstrate that the caspase-3 proenzyme has a mitochondrial and cytosolic distribution in nonapoptotic cells. The mitochondrial caspase-3 precursor is contained in the intermembrane space. Delivery of a variety of apoptotic stimuli is accompanied by loss of mitochondrial caspase-3 precursor staining and appearance of caspase-3 proteolytic activity. We propose that the mitochondrial subpopulation of caspase-3 precursor molecules is coupled to a distinct subset of apoptotic signaling pathways that are Bcl-2 sensitive and that are transduced through multiple mitochondrion-specific protein interactions.

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Figures

Figure 1

Figure 1

Characterization of anti–caspase-3 antiserum (R280). (a) 10-ng amounts of the purified large subunits (caspases 1–10, excluding caspase-6) and Ced-3 were immunoblotted with R280 antiserum. (b) Biotinylated recombinant mature caspase-3 was denatured by treatment with guanidium-HCl or SDS, as described in the Materials and Methods section. Native and denatured biotinylated caspase-3 were subsequently immunoprecipitated using R280, and the p17 subunit was detected by immunoblotting. (c) 46 μg of HUVEC lysate was immunoblotted with affinity-purified R280 antiserum. Similar results were obtained when keratinocyte lysates were immunoblotted with affinity-purified R280 antiserum (data not shown).

Figure 3

Figure 3

Biochemical analysis of mitochondrial caspase-3 precursor. (a) Caspase-3 precursor is detected by immunoblotting in human liver mitochondria. 200-μg aliquots of mitochondria, prepared from human liver as described in Materials and Methods, were incubated for 1 h at 37°C in the presence of the following: no additions (lane 1), 0.2 μg granzyme B (lane 2), 1% NP-40 (lane 3), or 1% NP-40 and 0.2 μg granzyme B (lane 4). Equal aliquots of these samples were electrophoresed and immunoblotted with the R280 anti–caspase-3 antibody. Note that the R280 antibody does not blot the 17-kD active enzyme well; the exposure shown in the figure was chosen to optimize visualization of the decreased 32-kD precursor caspase-3 (lane 4). The results shown are representative of three separate experiments. (b) Mitochondrial pro-caspase-3 colocalizes with adenylate kinase in the intermembrane space. Freshly isolated rat liver mitochondria were sub-fractionated by progressive digitonin treatment as described in Materials and Methods. Marker enzyme activities for the intermembrane space (adenylate kinase; circles) and matrix (fumarase; triangles) were measured in the resulting supernatants (solid symbols) and pellets (open symbols). Pro-caspase-3 (squares) was detected in the same fractions by immunoblotting using the R280 antibody that cross-reacts with rat pro-caspase-3. Marker enzyme and pro-caspase-3 distributions in supernatants and pellets are expressed as a percentage of the total recovered. (c) Distribution of caspase-3 precursor in HeLa cells. HeLa cells were fractionated into cytosolic (C) and mitochondrial (M) fractions as described in Materials and Methods. Equivalent volume amounts of cytosolic and mitochondrial fractions were electrophoresed, and relative amounts of caspase-3 precursor, cytochrome c, and cytochrome oxidase (subunit IV) were determined by immunoblotting. Results are representative of two separate experiments.

Figure 3

Figure 3

Biochemical analysis of mitochondrial caspase-3 precursor. (a) Caspase-3 precursor is detected by immunoblotting in human liver mitochondria. 200-μg aliquots of mitochondria, prepared from human liver as described in Materials and Methods, were incubated for 1 h at 37°C in the presence of the following: no additions (lane 1), 0.2 μg granzyme B (lane 2), 1% NP-40 (lane 3), or 1% NP-40 and 0.2 μg granzyme B (lane 4). Equal aliquots of these samples were electrophoresed and immunoblotted with the R280 anti–caspase-3 antibody. Note that the R280 antibody does not blot the 17-kD active enzyme well; the exposure shown in the figure was chosen to optimize visualization of the decreased 32-kD precursor caspase-3 (lane 4). The results shown are representative of three separate experiments. (b) Mitochondrial pro-caspase-3 colocalizes with adenylate kinase in the intermembrane space. Freshly isolated rat liver mitochondria were sub-fractionated by progressive digitonin treatment as described in Materials and Methods. Marker enzyme activities for the intermembrane space (adenylate kinase; circles) and matrix (fumarase; triangles) were measured in the resulting supernatants (solid symbols) and pellets (open symbols). Pro-caspase-3 (squares) was detected in the same fractions by immunoblotting using the R280 antibody that cross-reacts with rat pro-caspase-3. Marker enzyme and pro-caspase-3 distributions in supernatants and pellets are expressed as a percentage of the total recovered. (c) Distribution of caspase-3 precursor in HeLa cells. HeLa cells were fractionated into cytosolic (C) and mitochondrial (M) fractions as described in Materials and Methods. Equivalent volume amounts of cytosolic and mitochondrial fractions were electrophoresed, and relative amounts of caspase-3 precursor, cytochrome c, and cytochrome oxidase (subunit IV) were determined by immunoblotting. Results are representative of two separate experiments.

Figure 3

Figure 3

Biochemical analysis of mitochondrial caspase-3 precursor. (a) Caspase-3 precursor is detected by immunoblotting in human liver mitochondria. 200-μg aliquots of mitochondria, prepared from human liver as described in Materials and Methods, were incubated for 1 h at 37°C in the presence of the following: no additions (lane 1), 0.2 μg granzyme B (lane 2), 1% NP-40 (lane 3), or 1% NP-40 and 0.2 μg granzyme B (lane 4). Equal aliquots of these samples were electrophoresed and immunoblotted with the R280 anti–caspase-3 antibody. Note that the R280 antibody does not blot the 17-kD active enzyme well; the exposure shown in the figure was chosen to optimize visualization of the decreased 32-kD precursor caspase-3 (lane 4). The results shown are representative of three separate experiments. (b) Mitochondrial pro-caspase-3 colocalizes with adenylate kinase in the intermembrane space. Freshly isolated rat liver mitochondria were sub-fractionated by progressive digitonin treatment as described in Materials and Methods. Marker enzyme activities for the intermembrane space (adenylate kinase; circles) and matrix (fumarase; triangles) were measured in the resulting supernatants (solid symbols) and pellets (open symbols). Pro-caspase-3 (squares) was detected in the same fractions by immunoblotting using the R280 antibody that cross-reacts with rat pro-caspase-3. Marker enzyme and pro-caspase-3 distributions in supernatants and pellets are expressed as a percentage of the total recovered. (c) Distribution of caspase-3 precursor in HeLa cells. HeLa cells were fractionated into cytosolic (C) and mitochondrial (M) fractions as described in Materials and Methods. Equivalent volume amounts of cytosolic and mitochondrial fractions were electrophoresed, and relative amounts of caspase-3 precursor, cytochrome c, and cytochrome oxidase (subunit IV) were determined by immunoblotting. Results are representative of two separate experiments.

Figure 7

Figure 7

Time course of appearance of cleavage activity in keratinocyte lysates after UVB irradiation. (a) Whole cell lysates prepared at various times after UVB irradiation were assayed for cleavage of the fluorogenic substrate Ac-DEVD-AMC as described in the Materials and Methods section. Cleavage activity is expressed in U/mg, where a unit is defined as the amount of enzyme needed to produce 1 pmol per min using 100 μM Ac-DEVD-AMC. Four separate experiments yielded similar results. (b) PARP cleavage is observed in keratinocytes 6 h after UV irradiation. Irradiated and control nonirradiated keratinocytes were lysed at the indicated times as described in Materials and Methods. Whole cell lysates (lanes 1–3), as well as whole cell lysates incubated in vitro for 2 h at 37°C (lanes 4–6), were immunoblotted with a serum recognizing PARP (Casciola-Rosen et al., 1995). The migration positions of intact PARP (113 kD) and its cleavage fragment (89 kD) are marked. 60 μg of protein was loaded in each lane. This experiment was repeated on three separate occasions with similar results. Identical results were obtained using HeLa cells.

Figure 2

Figure 2

Caspase-3 is localized in the cytosol and mitochondria of keratinocytes and HUVECs. Keratinocytes (a–c) and HUVECs (d–f) were labeled with MitoTracker, fixed, permeabilized, and stained with affinity-purified polyclonal rabbit anti–caspase-3 precursor antibody (R280) as described in Materials and Methods. The stained cells were examined by confocal immunofluorescence microscopy. Caspase-3 antibodies were visualized with fluorescein-conjugated goat anti–rabbit IgG and assigned the color green (b and e), whereas mitochondria labeled with MitoTracker were assigned the color red (a and d). When red and green images were merged, overlapping red and green pixels appeared orange/yellow (c and f). The experiments were repeated on 11 (a–c) or 4 (d–f) separate occasions with identical results. Bar, 10 μm.

Figure 4

Figure 4

Immunolocalization of the caspase-3 precursor. Ultrathin sections of cultured WIF-B cells were labeled with affinity-purified R280 antibodies and 12-nm colloidal gold conjugated to donkey anti–rabbit IgG. a–d show caspase-3 precursor staining within mitochondria. Gold particles were localized to just inside the outer membrane (a and c), as well as to inner membrane cristae (a, b, and d). Omission of primary antibody resulted in the complete abolition of both cytosolic and mitochondrial staining (data not shown). Similar results were obtained in HeLa cells (data not shown). Bar, 300 nm.

Figure 5

Figure 5

Lack of mitochondrial caspase-3 precursor staining in keratinocytes induced to become apoptotic by UVB irradiation. Control nonirradiated keratinocytes (a–c) or keratinocytes UVB-irradiated and subsequently incubated for 8 h (d–f) were labeled with MitoTracker and then fixed, permeabilized, and stained with R280. Mitochondrial staining was visualized in red (a and d), whereas pro-caspase-3 staining was visualized in green (b and e). When images were merged (c and f), overlapping red and green pixels appeared orange/yellow. Arrowheads denote apoptotic surface blebs. Experiments were repeated on 10 separate occasions with similar results. Bar, 10 μm.

Figure 6

Figure 6

Lack of mitochondrial caspase-3 precursor staining and MitoTracker labeling in staurosporine-induced apoptosis. Keratinocytes were incubated with 5 μM staurosporine for 4.5 h before MitoTracker labeling (c) and double-staining with DAPI (a) and affinity-purified R280 antibodies (b). (a) DAPI staining of nuclei. Arrowheads denote fragmented, condensed nuclei typical of apoptotic cells; the arrow marks a normal, nonapoptotic nucleus. (b) R280 staining of pro-caspase-3. Caspase-3 precursor stains in normal cells (arrow) but is absent in apoptotic cells (arrowhead). (c) MitoTracker staining of mitochondria. Mitochondria are labeled with MitoTracker in cells with a normal nucleus (arrow) but do not label in cells with apoptotic nuclei (arrowhead). (a–c) These results are representative of three separate experiments. Bar, 10 μm.

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