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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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GCN-2 Acts in a Complementary Protective Pathway to that of ATFS-1 and the Induction of Mitochondrial Chaperone Genes.(A) Immunoblot of phosphorylated eIF2α from wild-type, clk-1(qm30), clk-1(qm30);gcn-2(ok871), clk-1(qm30);haf-1(ok705) animals fed vector(RNAi) and clk-1(qm30) animals fed atfs-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and raised from eggs on the indicated RNAi plate and harvested at the L4 stage. (B) Quantification of developmental rates of gcn-2(ok871), atfs-1(tm4525) and atfs-1(tm4525);gcn-2(ok871) animals. Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (C) Quantification of developmental rates of atfs-1(tm4525) and atfs-1(tm4525);gcn-2(ok871) animals raised on vector(RNAi) or spg-7(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (D) Quantification of developmental rates of clk-1(qm30) and clk-1(qm30);gcn-2(ok871) animals raised on vector(RNAi) or atfs-1(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 6. (E) Lifespan analysis of wild-type (median lifespan 20.0 days) and atfs-1(tm4525);gcn-2(ok871) animals (median lifespan 18.0 days); p = 0.3230, log-rank test. (F) Scheme of the hypothesized relationship of the two branches of the UPRmt where HAF-1 and ATFS-1 regulate mitochondrial chaperone gene induction and GCN-2 phosphorylates eIF2α to attenuate protein translation in response to mitochondrial stress.
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pgen-1002760-g008: GCN-2 Acts in a Complementary Protective Pathway to that of ATFS-1 and the Induction of Mitochondrial Chaperone Genes.(A) Immunoblot of phosphorylated eIF2α from wild-type, clk-1(qm30), clk-1(qm30);gcn-2(ok871), clk-1(qm30);haf-1(ok705) animals fed vector(RNAi) and clk-1(qm30) animals fed atfs-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and raised from eggs on the indicated RNAi plate and harvested at the L4 stage. (B) Quantification of developmental rates of gcn-2(ok871), atfs-1(tm4525) and atfs-1(tm4525);gcn-2(ok871) animals. Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (C) Quantification of developmental rates of atfs-1(tm4525) and atfs-1(tm4525);gcn-2(ok871) animals raised on vector(RNAi) or spg-7(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (D) Quantification of developmental rates of clk-1(qm30) and clk-1(qm30);gcn-2(ok871) animals raised on vector(RNAi) or atfs-1(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 6. (E) Lifespan analysis of wild-type (median lifespan 20.0 days) and atfs-1(tm4525);gcn-2(ok871) animals (median lifespan 18.0 days); p = 0.3230, log-rank test. (F) Scheme of the hypothesized relationship of the two branches of the UPRmt where HAF-1 and ATFS-1 regulate mitochondrial chaperone gene induction and GCN-2 phosphorylates eIF2α to attenuate protein translation in response to mitochondrial stress.

Mentions: The above data are consistent with GCN-2-dependent eIF2α phosphorylation and translation attenuation playing a protective role in maintaining mitochondrial function similar to the protection provided by the induced mitochondrial chaperone expression regulated by HAF-1 and ATFS-1 [11]. Therefore, we sought to determine the potential interaction or relationship between GCN-2 and ATFS-1/HAF-1. If they act in complementary pathways, we hypothesized that loss-of-function of both should be more detrimental than loss of either individual pathway. Inhibition of one pathway would cause more stress placing additional burden on the other pathway to maintain the mitochondrial protein-folding environment resulting in further activation of the complementary pathway. As indicated in Figure 2B, in the presence of stress, GCN-2 inhibition results in further activation of hsp-60pr::gfp expression. Similarly, reducing eIF2α phosphorylation by inhibiting ROS accumulation resulted in increased activation of hsp-60pr::gfp activation by ATFS-1 (Figure 7A). To determine if inhibition of mitochondrial chaperone induction during stress caused a further upregulation of the GCN-2 pathway and an increase in eIF2α phosphorylation, we examined phospo-eIF2α levels in clk-1(qm30) animals lacking HAF-1 or ATFS-1. clk-1(qm30) animals displayed an increase in eIF2α phosphorylation which was further increased in combination with the haf-1(ok705) deletion or when fed atfs-1(RNAi) (Figure 8A), consistent with GCN-2 acting in a separate and complementary pathway to that of ATFS-1 and HAF-1.


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)

GCN-2 Acts in a Complementary Protective Pathway to that of ATFS-1 and the Induction of Mitochondrial Chaperone Genes.(A) Immunoblot of phosphorylated eIF2α from wild-type, clk-1(qm30), clk-1(qm30);gcn-2(ok871), clk-1(qm30);haf-1(ok705) animals fed vector(RNAi) and clk-1(qm30) animals fed atfs-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and raised from eggs on the indicated RNAi plate and harvested at the L4 stage. (B) Quantification of developmental rates of gcn-2(ok871), atfs-1(tm4525) and atfs-1(tm4525);gcn-2(ok871) animals. Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (C) Quantification of developmental rates of atfs-1(tm4525) and atfs-1(tm4525);gcn-2(ok871) animals raised on vector(RNAi) or spg-7(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (D) Quantification of developmental rates of clk-1(qm30) and clk-1(qm30);gcn-2(ok871) animals raised on vector(RNAi) or atfs-1(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 6. (E) Lifespan analysis of wild-type (median lifespan 20.0 days) and atfs-1(tm4525);gcn-2(ok871) animals (median lifespan 18.0 days); p = 0.3230, log-rank test. (F) Scheme of the hypothesized relationship of the two branches of the UPRmt where HAF-1 and ATFS-1 regulate mitochondrial chaperone gene induction and GCN-2 phosphorylates eIF2α to attenuate protein translation in response to mitochondrial stress.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375257&req=5

pgen-1002760-g008: GCN-2 Acts in a Complementary Protective Pathway to that of ATFS-1 and the Induction of Mitochondrial Chaperone Genes.(A) Immunoblot of phosphorylated eIF2α from wild-type, clk-1(qm30), clk-1(qm30);gcn-2(ok871), clk-1(qm30);haf-1(ok705) animals fed vector(RNAi) and clk-1(qm30) animals fed atfs-1(RNAi). The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and raised from eggs on the indicated RNAi plate and harvested at the L4 stage. (B) Quantification of developmental rates of gcn-2(ok871), atfs-1(tm4525) and atfs-1(tm4525);gcn-2(ok871) animals. Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (C) Quantification of developmental rates of atfs-1(tm4525) and atfs-1(tm4525);gcn-2(ok871) animals raised on vector(RNAi) or spg-7(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (D) Quantification of developmental rates of clk-1(qm30) and clk-1(qm30);gcn-2(ok871) animals raised on vector(RNAi) or atfs-1(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 6. (E) Lifespan analysis of wild-type (median lifespan 20.0 days) and atfs-1(tm4525);gcn-2(ok871) animals (median lifespan 18.0 days); p = 0.3230, log-rank test. (F) Scheme of the hypothesized relationship of the two branches of the UPRmt where HAF-1 and ATFS-1 regulate mitochondrial chaperone gene induction and GCN-2 phosphorylates eIF2α to attenuate protein translation in response to mitochondrial stress.
Mentions: The above data are consistent with GCN-2-dependent eIF2α phosphorylation and translation attenuation playing a protective role in maintaining mitochondrial function similar to the protection provided by the induced mitochondrial chaperone expression regulated by HAF-1 and ATFS-1 [11]. Therefore, we sought to determine the potential interaction or relationship between GCN-2 and ATFS-1/HAF-1. If they act in complementary pathways, we hypothesized that loss-of-function of both should be more detrimental than loss of either individual pathway. Inhibition of one pathway would cause more stress placing additional burden on the other pathway to maintain the mitochondrial protein-folding environment resulting in further activation of the complementary pathway. As indicated in Figure 2B, in the presence of stress, GCN-2 inhibition results in further activation of hsp-60pr::gfp expression. Similarly, reducing eIF2α phosphorylation by inhibiting ROS accumulation resulted in increased activation of hsp-60pr::gfp activation by ATFS-1 (Figure 7A). To determine if inhibition of mitochondrial chaperone induction during stress caused a further upregulation of the GCN-2 pathway and an increase in eIF2α phosphorylation, we examined phospo-eIF2α levels in clk-1(qm30) animals lacking HAF-1 or ATFS-1. clk-1(qm30) animals displayed an increase in eIF2α phosphorylation which was further increased in combination with the haf-1(ok705) deletion or when fed atfs-1(RNAi) (Figure 8A), consistent with GCN-2 acting in a separate and complementary pathway to that of ATFS-1 and HAF-1.

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