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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 Is Required for Development and Mitochondrial Maintenance during Mitochondrial Stress.(A) Quantification of developmental rates of isp-1(qm150) and isp-1(qm150);gcn-2(ok871) animals. Synchronized worms were raised from eggs and animals of different developmental stages were scored and plotted as percent of total animals on day 6. (B) Developmental rates of clk-1(qm30) and clk-1(qm30);gcn-2(ok871) worms quantified as in (A) on day 5. (C) Wild-type and gcn-2(ok871) animals were raised on plates containing 1 µM rotenone. Rates of development were quantified on day 3. (D) Rates of oxygen consumption of synchronized wild-type or gcn-2(ok871) animals at the L4 stage. Shown is the mean ± SEM oxygen consumption normalized to protein content (n = 3). (E) Oxygen consumption rates of synchronized wild-type, clk-1(qm30) or clk-1(qm30);gcn-2(ok871) worms at the L4 stage. Shown is the mean ± SEM oxygen consumption normalized to protein content (n = 3, *p<0.05). (F) Immunoblots of lysates from wild-type, clk-1(qm30) or clk-1(qm30);gcn-2(ok871) probed with anti-DNP antibody (see Materials and Methods). The anti-HDEL immunoblot serves as a loading control. (G) Representative fluorescent photomicrographs of body wall muscle cells in transgenic animals expressing mitochondria-targeted GFP (myo-3pr::GFPmt) fed vector or gcn-2(RNAi). (H) Plot of the number of body strokes per minute (thrashing assay) of wild-type or myo-3pr::gfpmt transgenic animals raised on vector or gcn-2(RNAi). Shown is the mean±SEM obtained by counting strokes/min of 3-day-old animals (n = 5, *p<0.05).
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pgen-1002760-g005: GCN-2 Is Required for Development and Mitochondrial Maintenance during Mitochondrial Stress.(A) Quantification of developmental rates of isp-1(qm150) and isp-1(qm150);gcn-2(ok871) animals. Synchronized worms were raised from eggs and animals of different developmental stages were scored and plotted as percent of total animals on day 6. (B) Developmental rates of clk-1(qm30) and clk-1(qm30);gcn-2(ok871) worms quantified as in (A) on day 5. (C) Wild-type and gcn-2(ok871) animals were raised on plates containing 1 µM rotenone. Rates of development were quantified on day 3. (D) Rates of oxygen consumption of synchronized wild-type or gcn-2(ok871) animals at the L4 stage. Shown is the mean ± SEM oxygen consumption normalized to protein content (n = 3). (E) Oxygen consumption rates of synchronized wild-type, clk-1(qm30) or clk-1(qm30);gcn-2(ok871) worms at the L4 stage. Shown is the mean ± SEM oxygen consumption normalized to protein content (n = 3, *p<0.05). (F) Immunoblots of lysates from wild-type, clk-1(qm30) or clk-1(qm30);gcn-2(ok871) probed with anti-DNP antibody (see Materials and Methods). The anti-HDEL immunoblot serves as a loading control. (G) Representative fluorescent photomicrographs of body wall muscle cells in transgenic animals expressing mitochondria-targeted GFP (myo-3pr::GFPmt) fed vector or gcn-2(RNAi). (H) Plot of the number of body strokes per minute (thrashing assay) of wild-type or myo-3pr::gfpmt transgenic animals raised on vector or gcn-2(RNAi). Shown is the mean±SEM obtained by counting strokes/min of 3-day-old animals (n = 5, *p<0.05).

Mentions: As our data indicated that GCN-2 phosphorylates eIF2α in response to mitochondrial stress, we sought to determine the role of GCN-2 in development and mitochondrial maintenance during mitochondrial stress. gcn-2 deletion or RNAi had no observable effect on worm development in the absence of stress (Figure S4). However, in the presence of mitochondrial stress caused by either the isp-1(qm150) or clk-1(qm30) mutations, gcn-2 deletion significantly slowed development (Figure 5A and 5B). Furthermore, exposure to the NADH ubiquinone oxidoreductase (complex I) inhibitor rotenone or spg-7(RNAi) also significantly delayed development of gcn-2(ok871) worms relative to wild-type worms (Figure 5C and data not shown) indicating a protective role for GCN-2 during mitochondrial stress.


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 Is Required for Development and Mitochondrial Maintenance during Mitochondrial Stress.(A) Quantification of developmental rates of isp-1(qm150) and isp-1(qm150);gcn-2(ok871) animals. Synchronized worms were raised from eggs and animals of different developmental stages were scored and plotted as percent of total animals on day 6. (B) Developmental rates of clk-1(qm30) and clk-1(qm30);gcn-2(ok871) worms quantified as in (A) on day 5. (C) Wild-type and gcn-2(ok871) animals were raised on plates containing 1 µM rotenone. Rates of development were quantified on day 3. (D) Rates of oxygen consumption of synchronized wild-type or gcn-2(ok871) animals at the L4 stage. Shown is the mean ± SEM oxygen consumption normalized to protein content (n = 3). (E) Oxygen consumption rates of synchronized wild-type, clk-1(qm30) or clk-1(qm30);gcn-2(ok871) worms at the L4 stage. Shown is the mean ± SEM oxygen consumption normalized to protein content (n = 3, *p<0.05). (F) Immunoblots of lysates from wild-type, clk-1(qm30) or clk-1(qm30);gcn-2(ok871) probed with anti-DNP antibody (see Materials and Methods). The anti-HDEL immunoblot serves as a loading control. (G) Representative fluorescent photomicrographs of body wall muscle cells in transgenic animals expressing mitochondria-targeted GFP (myo-3pr::GFPmt) fed vector or gcn-2(RNAi). (H) Plot of the number of body strokes per minute (thrashing assay) of wild-type or myo-3pr::gfpmt transgenic animals raised on vector or gcn-2(RNAi). Shown is the mean±SEM obtained by counting strokes/min of 3-day-old animals (n = 5, *p<0.05).
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Related In: Results  -  Collection

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pgen-1002760-g005: GCN-2 Is Required for Development and Mitochondrial Maintenance during Mitochondrial Stress.(A) Quantification of developmental rates of isp-1(qm150) and isp-1(qm150);gcn-2(ok871) animals. Synchronized worms were raised from eggs and animals of different developmental stages were scored and plotted as percent of total animals on day 6. (B) Developmental rates of clk-1(qm30) and clk-1(qm30);gcn-2(ok871) worms quantified as in (A) on day 5. (C) Wild-type and gcn-2(ok871) animals were raised on plates containing 1 µM rotenone. Rates of development were quantified on day 3. (D) Rates of oxygen consumption of synchronized wild-type or gcn-2(ok871) animals at the L4 stage. Shown is the mean ± SEM oxygen consumption normalized to protein content (n = 3). (E) Oxygen consumption rates of synchronized wild-type, clk-1(qm30) or clk-1(qm30);gcn-2(ok871) worms at the L4 stage. Shown is the mean ± SEM oxygen consumption normalized to protein content (n = 3, *p<0.05). (F) Immunoblots of lysates from wild-type, clk-1(qm30) or clk-1(qm30);gcn-2(ok871) probed with anti-DNP antibody (see Materials and Methods). The anti-HDEL immunoblot serves as a loading control. (G) Representative fluorescent photomicrographs of body wall muscle cells in transgenic animals expressing mitochondria-targeted GFP (myo-3pr::GFPmt) fed vector or gcn-2(RNAi). (H) Plot of the number of body strokes per minute (thrashing assay) of wild-type or myo-3pr::gfpmt transgenic animals raised on vector or gcn-2(RNAi). Shown is the mean±SEM obtained by counting strokes/min of 3-day-old animals (n = 5, *p<0.05).
Mentions: As our data indicated that GCN-2 phosphorylates eIF2α in response to mitochondrial stress, we sought to determine the role of GCN-2 in development and mitochondrial maintenance during mitochondrial stress. gcn-2 deletion or RNAi had no observable effect on worm development in the absence of stress (Figure S4). However, in the presence of mitochondrial stress caused by either the isp-1(qm150) or clk-1(qm30) mutations, gcn-2 deletion significantly slowed development (Figure 5A and 5B). Furthermore, exposure to the NADH ubiquinone oxidoreductase (complex I) inhibitor rotenone or spg-7(RNAi) also significantly delayed development of gcn-2(ok871) worms relative to wild-type worms (Figure 5C and data not shown) indicating a protective role for GCN-2 during mitochondrial stress.

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