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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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Phosphorylation of eIF2α during Mitochondrial Stress Requires GCN-2.(A) Immunoblot of phospho-eIF2α from wild-type, clk-1(qm30) and clk-1(qm30);gcn-2(ok871) animals fed vector or gsp-1(RNAi). The total eIF2α and anti-HDEL immunoblots serve as loading controls. Synchronized animals were raised from eggs and harvested at the L4 stage. (B) Immunoblot of phospho-eIF2α from wild-type, isp-1(qm150) and isp-1(qm150);gcn-2(ok871) animals fed vector or gsp-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on the indicated RNAi plates and harvested at the L4 stage. (C) Immunoblot of phospho-eIF2α from wild-type, clk-1(qm30), clk-1(qm30);gcn-2(ok871) or clk-1(qm30);pek-1(zcdf2) worms. The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on vector(RNAi) plates and harvested at the L4 stage.
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pgen-1002760-g004: Phosphorylation of eIF2α during Mitochondrial Stress Requires GCN-2.(A) Immunoblot of phospho-eIF2α from wild-type, clk-1(qm30) and clk-1(qm30);gcn-2(ok871) animals fed vector or gsp-1(RNAi). The total eIF2α and anti-HDEL immunoblots serve as loading controls. Synchronized animals were raised from eggs and harvested at the L4 stage. (B) Immunoblot of phospho-eIF2α from wild-type, isp-1(qm150) and isp-1(qm150);gcn-2(ok871) animals fed vector or gsp-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on the indicated RNAi plates and harvested at the L4 stage. (C) Immunoblot of phospho-eIF2α from wild-type, clk-1(qm30), clk-1(qm30);gcn-2(ok871) or clk-1(qm30);pek-1(zcdf2) worms. The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on vector(RNAi) plates and harvested at the L4 stage.

Mentions: The data presented above suggest that GCN-2 activity promotes mitochondrial protein folding during mitochondrial stress. Therefore, we hypothesized that eIF2α phosphorylation would increase in a GCN-2-dependent manner in response to mitochondrial dysfunction. Indeed, phospho-eIF2α levels were increased relative to total eIF2α protein levels in the clk-1(qm30)mutant, which was absent in the gcn-2(ok871) mutant strain (Figure 4A). In contrast, gsp-1(RNAi) caused a further increase in phospho-eIF2α levels (Figure 4A). A similar result was observed in the isp-1(qm150) mutant, supporting the role of GCN-2 in eIF2α phosphorylation in response to stress (Figure 4B). As gcn-2(RNAi) perturbs the mitochondrial protein folding environment and GSP-1 knockdown promotes mitochondrial protein homeostasis as indicated by reduced hsp-60pr::gfp expression (Figure 2B and Figure S2B), these data suggest a correlation between an increase in phospho-eIF2α and a more favorable mitochondrial protein-folding environment.


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)

Phosphorylation of eIF2α during Mitochondrial Stress Requires GCN-2.(A) Immunoblot of phospho-eIF2α from wild-type, clk-1(qm30) and clk-1(qm30);gcn-2(ok871) animals fed vector or gsp-1(RNAi). The total eIF2α and anti-HDEL immunoblots serve as loading controls. Synchronized animals were raised from eggs and harvested at the L4 stage. (B) Immunoblot of phospho-eIF2α from wild-type, isp-1(qm150) and isp-1(qm150);gcn-2(ok871) animals fed vector or gsp-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on the indicated RNAi plates and harvested at the L4 stage. (C) Immunoblot of phospho-eIF2α from wild-type, clk-1(qm30), clk-1(qm30);gcn-2(ok871) or clk-1(qm30);pek-1(zcdf2) worms. The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on vector(RNAi) plates 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-g004: Phosphorylation of eIF2α during Mitochondrial Stress Requires GCN-2.(A) Immunoblot of phospho-eIF2α from wild-type, clk-1(qm30) and clk-1(qm30);gcn-2(ok871) animals fed vector or gsp-1(RNAi). The total eIF2α and anti-HDEL immunoblots serve as loading controls. Synchronized animals were raised from eggs and harvested at the L4 stage. (B) Immunoblot of phospho-eIF2α from wild-type, isp-1(qm150) and isp-1(qm150);gcn-2(ok871) animals fed vector or gsp-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on the indicated RNAi plates and harvested at the L4 stage. (C) Immunoblot of phospho-eIF2α from wild-type, clk-1(qm30), clk-1(qm30);gcn-2(ok871) or clk-1(qm30);pek-1(zcdf2) worms. The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on vector(RNAi) plates and harvested at the L4 stage.
Mentions: The data presented above suggest that GCN-2 activity promotes mitochondrial protein folding during mitochondrial stress. Therefore, we hypothesized that eIF2α phosphorylation would increase in a GCN-2-dependent manner in response to mitochondrial dysfunction. Indeed, phospho-eIF2α levels were increased relative to total eIF2α protein levels in the clk-1(qm30)mutant, which was absent in the gcn-2(ok871) mutant strain (Figure 4A). In contrast, gsp-1(RNAi) caused a further increase in phospho-eIF2α levels (Figure 4A). A similar result was observed in the isp-1(qm150) mutant, supporting the role of GCN-2 in eIF2α phosphorylation in response to stress (Figure 4B). As gcn-2(RNAi) perturbs the mitochondrial protein folding environment and GSP-1 knockdown promotes mitochondrial protein homeostasis as indicated by reduced hsp-60pr::gfp expression (Figure 2B and Figure S2B), these data suggest a correlation between an increase in phospho-eIF2α and a more favorable mitochondrial protein-folding environment.

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