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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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Generation of mitochondrial- and the ER- targeted Bak mutants. (A) Schemes of the Bak mutants. The carboxy-terminal transmembrane domain of Bak was deleted (ΔC) or replaced with the mitochondria-targeting sequence of ActA (ActA) or the ER-targeting sequence of cb5. (B) Targeting the Bak mutants to mitochondria and the ER. Bak mutants were expressed in MCF7 cells. Mitochondria marker COX IV and the ER marker calnexin were visualized by immunofluorescence. Note that the Bak-ActA mutant colocalized with COX IV but not calnexin, and the Bak-cb5 mutant colocalized with calnexin but not COX IV.
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fig4: Generation of mitochondrial- and the ER- targeted Bak mutants. (A) Schemes of the Bak mutants. The carboxy-terminal transmembrane domain of Bak was deleted (ΔC) or replaced with the mitochondria-targeting sequence of ActA (ActA) or the ER-targeting sequence of cb5. (B) Targeting the Bak mutants to mitochondria and the ER. Bak mutants were expressed in MCF7 cells. Mitochondria marker COX IV and the ER marker calnexin were visualized by immunofluorescence. Note that the Bak-ActA mutant colocalized with COX IV but not calnexin, and the Bak-cb5 mutant colocalized with calnexin but not COX IV.

Mentions: To study the function of proapoptotic Bcl-2 proteins selectively expressed on different intracellular organelles, mutants containing either mitochondrial- or ER-targeting sequences were constructed. In these mutants, sequences of the listerial protein ActA, which specifically directs proteins to mitochondria, and that of cb5, which specifically targets proteins to the ER membrane, were used as previously described (Zhu et al., 1996). Although the carboxy-terminal hydrophobic transmembrane domain of Bak is well defined functionally, the membrane-targeting domain of Bax remains controversial. Therefore, we generated Bak mutants specifically targeted to mitochondria and the ER by replacing the carboxy-terminal transmembrane domain of Bak with the cb5 and ActA sequences (Fig. 4Figure 4.


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)

Generation of mitochondrial- and the ER- targeted Bak mutants. (A) Schemes of the Bak mutants. The carboxy-terminal transmembrane domain of Bak was deleted (ΔC) or replaced with the mitochondria-targeting sequence of ActA (ActA) or the ER-targeting sequence of cb5. (B) Targeting the Bak mutants to mitochondria and the ER. Bak mutants were expressed in MCF7 cells. Mitochondria marker COX IV and the ER marker calnexin were visualized by immunofluorescence. Note that the Bak-ActA mutant colocalized with COX IV but not calnexin, and the Bak-cb5 mutant colocalized with calnexin but not COX IV.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172724&req=5

fig4: Generation of mitochondrial- and the ER- targeted Bak mutants. (A) Schemes of the Bak mutants. The carboxy-terminal transmembrane domain of Bak was deleted (ΔC) or replaced with the mitochondria-targeting sequence of ActA (ActA) or the ER-targeting sequence of cb5. (B) Targeting the Bak mutants to mitochondria and the ER. Bak mutants were expressed in MCF7 cells. Mitochondria marker COX IV and the ER marker calnexin were visualized by immunofluorescence. Note that the Bak-ActA mutant colocalized with COX IV but not calnexin, and the Bak-cb5 mutant colocalized with calnexin but not COX IV.
Mentions: To study the function of proapoptotic Bcl-2 proteins selectively expressed on different intracellular organelles, mutants containing either mitochondrial- or ER-targeting sequences were constructed. In these mutants, sequences of the listerial protein ActA, which specifically directs proteins to mitochondria, and that of cb5, which specifically targets proteins to the ER membrane, were used as previously described (Zhu et al., 1996). Although the carboxy-terminal hydrophobic transmembrane domain of Bak is well defined functionally, the membrane-targeting domain of Bax remains controversial. Therefore, we generated Bak mutants specifically targeted to mitochondria and the ER by replacing the carboxy-terminal transmembrane domain of Bak with the cb5 and ActA sequences (Fig. 4Figure 4.

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