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Protective coupling of mitochondrial function and protein synthesis via the eIF2α kinase GCN-2.

Baker BM, Nargund AM, Sun T, Haynes CM - PLoS Genet. (2012)

Bottom Line: During mitochondrial dysfunction, GCN-2-dependent eIF2α phosphorylation is required for development as well as the lifespan extension observed in Caenorhabditis elegans.Reactive oxygen species (ROS) generated from dysfunctional mitochondria are required for GCN-2-dependent eIF2α phosphorylation but not ATFS-1 activation.These findings are consistent with translational control and stress-dependent chaperone induction acting in complementary arms of the UPR(mt).

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Cells respond to defects in mitochondrial function by activating signaling pathways that restore homeostasis. The mitochondrial peptide exporter HAF-1 and the bZip transcription factor ATFS-1 represent one stress response pathway that regulates the transcription of mitochondrial chaperone genes during mitochondrial dysfunction. Here, we report that GCN-2, an eIF2α kinase that modulates cytosolic protein synthesis, functions in a complementary pathway to that of HAF-1 and ATFS-1. During mitochondrial dysfunction, GCN-2-dependent eIF2α phosphorylation is required for development as well as the lifespan extension observed in Caenorhabditis elegans. Reactive oxygen species (ROS) generated from dysfunctional mitochondria are required for GCN-2-dependent eIF2α phosphorylation but not ATFS-1 activation. Simultaneous deletion of ATFS-1 and GCN-2 compounds the developmental defects associated with mitochondrial stress, while stressed animals lacking GCN-2 display a greater dependence on ATFS-1 and stronger induction of mitochondrial chaperone genes. These findings are consistent with translational control and stress-dependent chaperone induction acting in complementary arms of the UPR(mt).

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Knockdown of GCN-2 and GSP-1 Modulates eIF2α Phosphorylation Status and Mitochondrial Protein Homeostasis.(A) Fluorescent photomicrographs of hsp-4pr::gfp reporter animals raised on vector(RNAi), gcn-2(RNAi) or pek-1(RNAi). Worms were hatched on the individual RNAi plates and maintained at 20°C (upper panels) or subjected to heat shock at 30°C (3 hours) to induce ER stress (lower panels) at the L4 developmental stage. (B) Comparison of the amino acid sequence surrounding the conserved serine residue of eIF2α that is phosphorylated by the eIF2α kinases including GCN-2. (C) Immunoblot of wild-type worm lysates untreated or treated with calf intestinal phosphatase (CIP) and probed with an antibody specific to the phosphorylated form of eIF2α. The endogenous ER protein HDEL was detected with a monoclonal antibody (lower panel) and serves as a loading control. (D) Immunoblot of phosphorylated eIF2α from wild-type, gcn-2(ok871) and gcn-2(ok871);pek-1(zcdf2) animals. The anti-eIF2α and anti-HDEL immunoblots serve as loading controls. (E) Immunoblot of phosphorylated eIF2α from wild-type, gcn-2(ok871), pek-1(zcdf2) and gcn-2(ok871);pek-1(zcdf2) animals raised on vector or gsp-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Animals were raised from eggs on vector or gsp-1(RNAi) and harvested at the L4 stage.
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pgen-1002760-g003: Knockdown of GCN-2 and GSP-1 Modulates eIF2α Phosphorylation Status and Mitochondrial Protein Homeostasis.(A) Fluorescent photomicrographs of hsp-4pr::gfp reporter animals raised on vector(RNAi), gcn-2(RNAi) or pek-1(RNAi). Worms were hatched on the individual RNAi plates and maintained at 20°C (upper panels) or subjected to heat shock at 30°C (3 hours) to induce ER stress (lower panels) at the L4 developmental stage. (B) Comparison of the amino acid sequence surrounding the conserved serine residue of eIF2α that is phosphorylated by the eIF2α kinases including GCN-2. (C) Immunoblot of wild-type worm lysates untreated or treated with calf intestinal phosphatase (CIP) and probed with an antibody specific to the phosphorylated form of eIF2α. The endogenous ER protein HDEL was detected with a monoclonal antibody (lower panel) and serves as a loading control. (D) Immunoblot of phosphorylated eIF2α from wild-type, gcn-2(ok871) and gcn-2(ok871);pek-1(zcdf2) animals. The anti-eIF2α and anti-HDEL immunoblots serve as loading controls. (E) Immunoblot of phosphorylated eIF2α from wild-type, gcn-2(ok871), pek-1(zcdf2) and gcn-2(ok871);pek-1(zcdf2) animals raised on vector or gsp-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Animals were raised from eggs on vector or gsp-1(RNAi) and harvested at the L4 stage.

Mentions: In addition to CeTor, rsks-1 and cel-1, we identified components that are known to regulate translation initiation by modulating the phosphorylation status of the translation initiation factor eIF2α. RNAi-knockdown of the eIF2α kinase General Control Non-derepressible-2 (GCN-2) further increased hsp-60pr::gfp expression in clk-1(qm30) animals, suggesting a role for GCN-2 in promoting mitochondrial protein homeostasis or function (Figure 2A and 2B). The effect of gcn-2(RNAi) on hsp-60pr::gfp expression was not due to direct effects on GFP translation as gcn-2(RNAi) did not cause induction of the ER stress reporter hsp-4pr::gfp (Figure 3A) suggesting a specific role for GCN-2 in promoting mitochondrial protein homeostasis. In unstressed animals, gcn-2(RNAi) did not effect hsp-60pr::gfp expression, suggesting its primary role is during stress (Figure 2B). Contrary to gcn-2(RNAi), our RNAi screen identified gsp-1(RNAi), which reduced hsp-60pr::gfp expression in both the clk-1(qm30) and isp-1(qm150) strains (Figure 2A, 2B and Figure S2B). GSP-1 encodes a protein phosphatase (PP1) required for numerous cellular dephosphorylation events [32], [33] and is homologous to the yeast phosphatase required for eIF2α dephosphorylation [34].


Protective coupling of mitochondrial function and protein synthesis via the eIF2α kinase GCN-2.

Baker BM, Nargund AM, Sun T, Haynes CM - PLoS Genet. (2012)

Knockdown of GCN-2 and GSP-1 Modulates eIF2α Phosphorylation Status and Mitochondrial Protein Homeostasis.(A) Fluorescent photomicrographs of hsp-4pr::gfp reporter animals raised on vector(RNAi), gcn-2(RNAi) or pek-1(RNAi). Worms were hatched on the individual RNAi plates and maintained at 20°C (upper panels) or subjected to heat shock at 30°C (3 hours) to induce ER stress (lower panels) at the L4 developmental stage. (B) Comparison of the amino acid sequence surrounding the conserved serine residue of eIF2α that is phosphorylated by the eIF2α kinases including GCN-2. (C) Immunoblot of wild-type worm lysates untreated or treated with calf intestinal phosphatase (CIP) and probed with an antibody specific to the phosphorylated form of eIF2α. The endogenous ER protein HDEL was detected with a monoclonal antibody (lower panel) and serves as a loading control. (D) Immunoblot of phosphorylated eIF2α from wild-type, gcn-2(ok871) and gcn-2(ok871);pek-1(zcdf2) animals. The anti-eIF2α and anti-HDEL immunoblots serve as loading controls. (E) Immunoblot of phosphorylated eIF2α from wild-type, gcn-2(ok871), pek-1(zcdf2) and gcn-2(ok871);pek-1(zcdf2) animals raised on vector or gsp-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Animals were raised from eggs on vector or gsp-1(RNAi) and harvested at the L4 stage.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375257&req=5

pgen-1002760-g003: Knockdown of GCN-2 and GSP-1 Modulates eIF2α Phosphorylation Status and Mitochondrial Protein Homeostasis.(A) Fluorescent photomicrographs of hsp-4pr::gfp reporter animals raised on vector(RNAi), gcn-2(RNAi) or pek-1(RNAi). Worms were hatched on the individual RNAi plates and maintained at 20°C (upper panels) or subjected to heat shock at 30°C (3 hours) to induce ER stress (lower panels) at the L4 developmental stage. (B) Comparison of the amino acid sequence surrounding the conserved serine residue of eIF2α that is phosphorylated by the eIF2α kinases including GCN-2. (C) Immunoblot of wild-type worm lysates untreated or treated with calf intestinal phosphatase (CIP) and probed with an antibody specific to the phosphorylated form of eIF2α. The endogenous ER protein HDEL was detected with a monoclonal antibody (lower panel) and serves as a loading control. (D) Immunoblot of phosphorylated eIF2α from wild-type, gcn-2(ok871) and gcn-2(ok871);pek-1(zcdf2) animals. The anti-eIF2α and anti-HDEL immunoblots serve as loading controls. (E) Immunoblot of phosphorylated eIF2α from wild-type, gcn-2(ok871), pek-1(zcdf2) and gcn-2(ok871);pek-1(zcdf2) animals raised on vector or gsp-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Animals were raised from eggs on vector or gsp-1(RNAi) and harvested at the L4 stage.
Mentions: In addition to CeTor, rsks-1 and cel-1, we identified components that are known to regulate translation initiation by modulating the phosphorylation status of the translation initiation factor eIF2α. RNAi-knockdown of the eIF2α kinase General Control Non-derepressible-2 (GCN-2) further increased hsp-60pr::gfp expression in clk-1(qm30) animals, suggesting a role for GCN-2 in promoting mitochondrial protein homeostasis or function (Figure 2A and 2B). The effect of gcn-2(RNAi) on hsp-60pr::gfp expression was not due to direct effects on GFP translation as gcn-2(RNAi) did not cause induction of the ER stress reporter hsp-4pr::gfp (Figure 3A) suggesting a specific role for GCN-2 in promoting mitochondrial protein homeostasis. In unstressed animals, gcn-2(RNAi) did not effect hsp-60pr::gfp expression, suggesting its primary role is during stress (Figure 2B). Contrary to gcn-2(RNAi), our RNAi screen identified gsp-1(RNAi), which reduced hsp-60pr::gfp expression in both the clk-1(qm30) and isp-1(qm150) strains (Figure 2A, 2B and Figure S2B). GSP-1 encodes a protein phosphatase (PP1) required for numerous cellular dephosphorylation events [32], [33] and is homologous to the yeast phosphatase required for eIF2α dephosphorylation [34].

Bottom Line: During mitochondrial dysfunction, GCN-2-dependent eIF2α phosphorylation is required for development as well as the lifespan extension observed in Caenorhabditis elegans.Reactive oxygen species (ROS) generated from dysfunctional mitochondria are required for GCN-2-dependent eIF2α phosphorylation but not ATFS-1 activation.These findings are consistent with translational control and stress-dependent chaperone induction acting in complementary arms of the UPR(mt).

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Cells respond to defects in mitochondrial function by activating signaling pathways that restore homeostasis. The mitochondrial peptide exporter HAF-1 and the bZip transcription factor ATFS-1 represent one stress response pathway that regulates the transcription of mitochondrial chaperone genes during mitochondrial dysfunction. Here, we report that GCN-2, an eIF2α kinase that modulates cytosolic protein synthesis, functions in a complementary pathway to that of HAF-1 and ATFS-1. During mitochondrial dysfunction, GCN-2-dependent eIF2α phosphorylation is required for development as well as the lifespan extension observed in Caenorhabditis elegans. Reactive oxygen species (ROS) generated from dysfunctional mitochondria are required for GCN-2-dependent eIF2α phosphorylation but not ATFS-1 activation. Simultaneous deletion of ATFS-1 and GCN-2 compounds the developmental defects associated with mitochondrial stress, while stressed animals lacking GCN-2 display a greater dependence on ATFS-1 and stronger induction of mitochondrial chaperone genes. These findings are consistent with translational control and stress-dependent chaperone induction acting in complementary arms of the UPR(mt).

Show MeSH
Related in: MedlinePlus