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

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).

Figure 1. ATFS-1 Is Required for Mitochondrial Chaperone Induction and Development during Mitochondrial Stress.

(A) Fluorescent photomicrographs of wild-type and atfs-1(tm4525);hsp-60_pr::gfp transgenic worms raised on vector or spg-7(RNAi). (B) Representative fluorescent photomicrographs of hsp-60_pr::gfp transgenic worms harboring the clk-1(qm30) or isp-1(qm150) alleles raised on vector(RNAi). (C) Images of clk-1(qm30) animals raised on vector or atfs-1(RNAi). Worms were plated at the L4 stage, allowed to develop to adulthood and lay eggs for 16 hours. The images were obtained five days after hatching.

Figure 2. Identification of Kinases and Phosphatases That Affect Protein Synthesis Impact UPR^mt Activation.

(A) Phosphatase and kinases whose knockdown by RNAi either increased or decreased hsp-60_pr::gfp expression in clk-1(qm30) mutant worms. (B) Fluorescent photomicrographs of hsp-60_pr::gfp expression in wild-type and clk-1(qm30) animals raised on vector, gcn-2 or gsp-1(RNAi).

Figure 3. Knockdown of GCN-2 and GSP-1 Modulates eIF2α Phosphorylation Status and Mitochondrial Protein Homeostasis.

(A) Fluorescent photomicrographs of hsp-4_pr::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.

Figure 4. 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.

Figure 5. 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-3_pr::GFP^mt) fed vector or gcn-2(RNAi). (H) Plot of the number of body strokes per minute (thrashing assay) of wild-type or myo-3_pr::gfp^mt 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).

Figure 6. GCN-2 Is Required for the Lifespan Extension Associated with Mitochondrial Dysfunction.

(A) Lifespan analysis of clk-1(qm30) animals fed vector (median survival 27.0 days) or gcn-2(RNAi) (median survival 17.0 days); p<0.0001, log-rank test. (B) Lifespan analysis of wild-type animals fed vector(RNAi) (median survival 21.0 days) or gcn-2(RNAi) (median survival 20.0 days); p = 0.6019 log-rank test.

Figure 7. Phosphorylation of eIF2α during Mitochondrial Stress Requires ROS.

(A) Fluorescent photomicrographs of clk-1(qm30);hsp-60_pr::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.

Figure 8. 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 UPR^mt 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.