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Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis.

Zong WX, Li C, Hatzivassiliou G, Lindsten T, Yu QC, Yuan J, Thompson CB - J. Cell Biol. (2003)

Bottom Line: In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells.In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak.These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Abramson Cancer Center, 421 Curie Blvd., BRB II/III, 445, Philadelphia, PA 19104-6160, USA. craig@mail.med.upenn.edu

ABSTRACT
Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells. In bax-/-bak-/- cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

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The ER-targeted Bak-cb5 induces depletion of the ER Ca2+ pool. (A) Cells with ER-targeted Bak lack thapsigargin-releasable intracellular Ca2+. bax−/−bak−/− cells were infected with retroviruses encoding GFP, Bak-IRES-GFP, Bak-ActA-IRES-GFP, or Bak-cb5-IRES-GFP. 48 h after infection, cells were loaded with Indo-1, and the ER Ca2+ release was measured by flow cytometry. Propidium iodide was added to determine the cell viability. The Ca2+ flux traces were derived from gated live GFP-positive cells. The [Ca2+]i was calibrated from the measured Indo-1 fluorescence ratio as described in the Materials and methods. The arrowhead indicates the time of thapsigargin addition. (B) Expression of ER-targeted Bak depleted intracellular Ca2+ storage in the ER. At the start of measurement, extracellular free Ca2+ concentration was reduced to zero. The arrowhead indicates the time when the extracellular free Ca2+ concentration was brought back to 2 mM. [Ca2+]i was analyzed as in A. (C and D) bax−/−bak−/− cells were infected with the Bak-cb5-IRES-GFP. (C) The measured Indo-1 fluorescence ratio between λ405 and λ475 is presented as an index of change in cytosolic [Ca2+] over time and presented separately for gated live GFP-positive (expressing Bak-cb5) or GFP-negative (not expressing Bak-cb5) cells. The absolute [Ca2+]i in the linear range is indicated on the right. The arrowhead indicates the time of thapsigargin addition. The arrow indicates the time of the addition of 8 mM CaCl2 to the medium. (D) Mitochondrial potential in GFP-positive (expressing Bak-cb5, green) and in GFP-negative (not expressing Bak-cb5, black) cells was measured by tetramethyl rhodamine ethyl ester (TMRE) staining.
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fig5: The ER-targeted Bak-cb5 induces depletion of the ER Ca2+ pool. (A) Cells with ER-targeted Bak lack thapsigargin-releasable intracellular Ca2+. bax−/−bak−/− cells were infected with retroviruses encoding GFP, Bak-IRES-GFP, Bak-ActA-IRES-GFP, or Bak-cb5-IRES-GFP. 48 h after infection, cells were loaded with Indo-1, and the ER Ca2+ release was measured by flow cytometry. Propidium iodide was added to determine the cell viability. The Ca2+ flux traces were derived from gated live GFP-positive cells. The [Ca2+]i was calibrated from the measured Indo-1 fluorescence ratio as described in the Materials and methods. The arrowhead indicates the time of thapsigargin addition. (B) Expression of ER-targeted Bak depleted intracellular Ca2+ storage in the ER. At the start of measurement, extracellular free Ca2+ concentration was reduced to zero. The arrowhead indicates the time when the extracellular free Ca2+ concentration was brought back to 2 mM. [Ca2+]i was analyzed as in A. (C and D) bax−/−bak−/− cells were infected with the Bak-cb5-IRES-GFP. (C) The measured Indo-1 fluorescence ratio between λ405 and λ475 is presented as an index of change in cytosolic [Ca2+] over time and presented separately for gated live GFP-positive (expressing Bak-cb5) or GFP-negative (not expressing Bak-cb5) cells. The absolute [Ca2+]i in the linear range is indicated on the right. The arrowhead indicates the time of thapsigargin addition. The arrow indicates the time of the addition of 8 mM CaCl2 to the medium. (D) Mitochondrial potential in GFP-positive (expressing Bak-cb5, green) and in GFP-negative (not expressing Bak-cb5, black) cells was measured by tetramethyl rhodamine ethyl ester (TMRE) staining.

Mentions: To determine whether the ER-targeted Bak or any of the other Bak mutants can perturb the integrity of the ER membrane, we determined the effects of these mutants on the ER Ca2+ store by assessing thapsigargin-induced ER Ca2+ release. bax−/−bak−/− cells were infected with retroviruses encoding GFP alone, or in combination with wild-type Bak, Bak-ActA, or Bak-cb5, and analyzed for changes in intracellular [Ca2+] after thapsigargin treatment. The thapsigargin-releasable pool of ER Ca2+ was depleted in cells expressing Bak-cb5 (Fig. 5Figure 5.


Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis.

Zong WX, Li C, Hatzivassiliou G, Lindsten T, Yu QC, Yuan J, Thompson CB - J. Cell Biol. (2003)

The ER-targeted Bak-cb5 induces depletion of the ER Ca2+ pool. (A) Cells with ER-targeted Bak lack thapsigargin-releasable intracellular Ca2+. bax−/−bak−/− cells were infected with retroviruses encoding GFP, Bak-IRES-GFP, Bak-ActA-IRES-GFP, or Bak-cb5-IRES-GFP. 48 h after infection, cells were loaded with Indo-1, and the ER Ca2+ release was measured by flow cytometry. Propidium iodide was added to determine the cell viability. The Ca2+ flux traces were derived from gated live GFP-positive cells. The [Ca2+]i was calibrated from the measured Indo-1 fluorescence ratio as described in the Materials and methods. The arrowhead indicates the time of thapsigargin addition. (B) Expression of ER-targeted Bak depleted intracellular Ca2+ storage in the ER. At the start of measurement, extracellular free Ca2+ concentration was reduced to zero. The arrowhead indicates the time when the extracellular free Ca2+ concentration was brought back to 2 mM. [Ca2+]i was analyzed as in A. (C and D) bax−/−bak−/− cells were infected with the Bak-cb5-IRES-GFP. (C) The measured Indo-1 fluorescence ratio between λ405 and λ475 is presented as an index of change in cytosolic [Ca2+] over time and presented separately for gated live GFP-positive (expressing Bak-cb5) or GFP-negative (not expressing Bak-cb5) cells. The absolute [Ca2+]i in the linear range is indicated on the right. The arrowhead indicates the time of thapsigargin addition. The arrow indicates the time of the addition of 8 mM CaCl2 to the medium. (D) Mitochondrial potential in GFP-positive (expressing Bak-cb5, green) and in GFP-negative (not expressing Bak-cb5, black) cells was measured by tetramethyl rhodamine ethyl ester (TMRE) staining.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172724&req=5

fig5: The ER-targeted Bak-cb5 induces depletion of the ER Ca2+ pool. (A) Cells with ER-targeted Bak lack thapsigargin-releasable intracellular Ca2+. bax−/−bak−/− cells were infected with retroviruses encoding GFP, Bak-IRES-GFP, Bak-ActA-IRES-GFP, or Bak-cb5-IRES-GFP. 48 h after infection, cells were loaded with Indo-1, and the ER Ca2+ release was measured by flow cytometry. Propidium iodide was added to determine the cell viability. The Ca2+ flux traces were derived from gated live GFP-positive cells. The [Ca2+]i was calibrated from the measured Indo-1 fluorescence ratio as described in the Materials and methods. The arrowhead indicates the time of thapsigargin addition. (B) Expression of ER-targeted Bak depleted intracellular Ca2+ storage in the ER. At the start of measurement, extracellular free Ca2+ concentration was reduced to zero. The arrowhead indicates the time when the extracellular free Ca2+ concentration was brought back to 2 mM. [Ca2+]i was analyzed as in A. (C and D) bax−/−bak−/− cells were infected with the Bak-cb5-IRES-GFP. (C) The measured Indo-1 fluorescence ratio between λ405 and λ475 is presented as an index of change in cytosolic [Ca2+] over time and presented separately for gated live GFP-positive (expressing Bak-cb5) or GFP-negative (not expressing Bak-cb5) cells. The absolute [Ca2+]i in the linear range is indicated on the right. The arrowhead indicates the time of thapsigargin addition. The arrow indicates the time of the addition of 8 mM CaCl2 to the medium. (D) Mitochondrial potential in GFP-positive (expressing Bak-cb5, green) and in GFP-negative (not expressing Bak-cb5, black) cells was measured by tetramethyl rhodamine ethyl ester (TMRE) staining.
Mentions: To determine whether the ER-targeted Bak or any of the other Bak mutants can perturb the integrity of the ER membrane, we determined the effects of these mutants on the ER Ca2+ store by assessing thapsigargin-induced ER Ca2+ release. bax−/−bak−/− cells were infected with retroviruses encoding GFP alone, or in combination with wild-type Bak, Bak-ActA, or Bak-cb5, and analyzed for changes in intracellular [Ca2+] after thapsigargin treatment. The thapsigargin-releasable pool of ER Ca2+ was depleted in cells expressing Bak-cb5 (Fig. 5Figure 5.

Bottom Line: In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells.In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak.These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Abramson Cancer Center, 421 Curie Blvd., BRB II/III, 445, Philadelphia, PA 19104-6160, USA. craig@mail.med.upenn.edu

ABSTRACT
Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells. In bax-/-bak-/- cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

Show MeSH
Related in: MedlinePlus