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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 ROS.(A) Fluorescent photomicrographs of clk-1(qm30);hsp-60pr::gfp animals synchronized and raised on control or plates containing 8 mM ascorbate. Images were obtained on day 5. (B) Immunoblot of phosphorylated eIF2α from clk-1(qm30) and isp-1(qm150) mutant worms untreated or treated with 25 mM ascorbate. The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and allowed to develop to adulthood, at which time they were treated with 25 mM ascorbate for 16 hours prior to harvest. (C) Immunoblot of phosphorylated eIF2α from wild-type and gcn-2(ok871) animals treated with 1 mM paraquat (PQ). The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and raised in liquid culture to the young adult stage when 1 mM PQ was added for 16 hours prior to harvest.
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pgen-1002760-g007: Phosphorylation of eIF2α during Mitochondrial Stress Requires ROS.(A) Fluorescent photomicrographs of clk-1(qm30);hsp-60pr::gfp animals synchronized and raised on control or plates containing 8 mM ascorbate. Images were obtained on day 5. (B) Immunoblot of phosphorylated eIF2α from clk-1(qm30) and isp-1(qm150) mutant worms untreated or treated with 25 mM ascorbate. The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and allowed to develop to adulthood, at which time they were treated with 25 mM ascorbate for 16 hours prior to harvest. (C) Immunoblot of phosphorylated eIF2α from wild-type and gcn-2(ok871) animals treated with 1 mM paraquat (PQ). The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and raised in liquid culture to the young adult stage when 1 mM PQ was added for 16 hours prior to harvest.

Mentions: We next sought to determine how phospho-eIF2α status is linked to mitochondrial dysfunction. While the most well-studied mechanism of GCN-2 activation is through starvation or amino acid depletion, hydrogen peroxide exposure also stimulates GCN-2 activity through a mechanism that requires the tRNA synthetase domain [52], [53]. Because clk-1(qm30) and isp-1(qm150) worms produce increased levels of ROS (Figure 5F) that are also required for their extended longevity [22], [23], we hypothesized that ROS generated from dysfunctional mitochondria act as an upstream signaling molecule coupling mitochondrial dysfunction to GCN-2 activation. If ROS are required for the observed increase in eIF2α phosphorylation during mitochondrial stress, then treatment with ROS scavengers would phenocopy GCN-2 inhibition with respect to hsp-60pr::gfp activation and the reduced accumulation of phospho-eIF2α in the presence of mitochondrial stress. Impressively, incubation of clk-1(qm30) animals with the ROS scavenger ascorbate resulted in increased hsp-60pr::gfp activation, similar to gcn-2(RNAi) (Figure 7A and Figure 2B). Ascorbate had no effect on the induction of hsp-60pr::gfp in unstressed animals (data not shown) as observed with gcn-2(RNAi) (Figure 2B). We next examined the impact of ascorbate on eIF2α phosphorylation in clk-1(qm30) and isp-1(qm150) animals. Ascorbate treatment, like GCN-2 inhibition, caused a reduction of eIF2α phosphorylation in both mutants supporting a role for ROS in GCN-2 signaling during mitochondrial stress (Figure 7B).


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 ROS.(A) Fluorescent photomicrographs of clk-1(qm30);hsp-60pr::gfp animals synchronized and raised on control or plates containing 8 mM ascorbate. Images were obtained on day 5. (B) Immunoblot of phosphorylated eIF2α from clk-1(qm30) and isp-1(qm150) mutant worms untreated or treated with 25 mM ascorbate. The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and allowed to develop to adulthood, at which time they were treated with 25 mM ascorbate for 16 hours prior to harvest. (C) Immunoblot of phosphorylated eIF2α from wild-type and gcn-2(ok871) animals treated with 1 mM paraquat (PQ). The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and raised in liquid culture to the young adult stage when 1 mM PQ was added for 16 hours prior to harvest.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375257&req=5

pgen-1002760-g007: Phosphorylation of eIF2α during Mitochondrial Stress Requires ROS.(A) Fluorescent photomicrographs of clk-1(qm30);hsp-60pr::gfp animals synchronized and raised on control or plates containing 8 mM ascorbate. Images were obtained on day 5. (B) Immunoblot of phosphorylated eIF2α from clk-1(qm30) and isp-1(qm150) mutant worms untreated or treated with 25 mM ascorbate. The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and allowed to develop to adulthood, at which time they were treated with 25 mM ascorbate for 16 hours prior to harvest. (C) Immunoblot of phosphorylated eIF2α from wild-type and gcn-2(ok871) animals treated with 1 mM paraquat (PQ). The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and raised in liquid culture to the young adult stage when 1 mM PQ was added for 16 hours prior to harvest.
Mentions: We next sought to determine how phospho-eIF2α status is linked to mitochondrial dysfunction. While the most well-studied mechanism of GCN-2 activation is through starvation or amino acid depletion, hydrogen peroxide exposure also stimulates GCN-2 activity through a mechanism that requires the tRNA synthetase domain [52], [53]. Because clk-1(qm30) and isp-1(qm150) worms produce increased levels of ROS (Figure 5F) that are also required for their extended longevity [22], [23], we hypothesized that ROS generated from dysfunctional mitochondria act as an upstream signaling molecule coupling mitochondrial dysfunction to GCN-2 activation. If ROS are required for the observed increase in eIF2α phosphorylation during mitochondrial stress, then treatment with ROS scavengers would phenocopy GCN-2 inhibition with respect to hsp-60pr::gfp activation and the reduced accumulation of phospho-eIF2α in the presence of mitochondrial stress. Impressively, incubation of clk-1(qm30) animals with the ROS scavenger ascorbate resulted in increased hsp-60pr::gfp activation, similar to gcn-2(RNAi) (Figure 7A and Figure 2B). Ascorbate had no effect on the induction of hsp-60pr::gfp in unstressed animals (data not shown) as observed with gcn-2(RNAi) (Figure 2B). We next examined the impact of ascorbate on eIF2α phosphorylation in clk-1(qm30) and isp-1(qm150) animals. Ascorbate treatment, like GCN-2 inhibition, caused a reduction of eIF2α phosphorylation in both mutants supporting a role for ROS in GCN-2 signaling during mitochondrial stress (Figure 7B).

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