Protective Coupling of Mitochondrial Function and Protein Synthesis via the eIF2α Kinase GCN-2

Mitochondrial Chaperone Induction by ATFS-1 Is Required for Development during Mitochondrial Stress

We have previously described a mitochondrial stress response pathway that upregulates mitochondrial chaperone genes in response to multiple perturbations in mitochondrial function [8], [11], [24]. RNAi experiments indicated a requirement for the bZip transcription factor ATFS-1 in mitochondrial chaperone induction as demonstrated by quantitative PCR experiments as well as using reporter strains where the hsp-60 promoter regulates expression of GFP (hsp-60_pr::gfp) [11]. In order to corroborate the requirement for ATFS-1, we obtained the atfs-1(tm4525) deletion strain which lacks 432 base pairs and most of exons 2–4, and crossed it into the reporter strain. Unlike wild-type worms, atfs-1(tm4525) animals were unable to induce hsp-60_pr::gfp when raised on spg-7(RNAi), a mitochondrial protease required for ETC quality control and mitochondrial ribosome biogenesis [25]. These results confirm the requirement for ATFS-1 in stress-induced mitochondrial chaperone gene induction (Figure 1A).

We next investigated ATFS-1-dependent hsp-60_pr::gfp activation in strains harboring the well-characterized clk-1(qm30) or isp-1(qm150) mutations [26], [27]. clk-1 encodes a mitochondrial protein required for ubiquinone synthesis [28], which acts as a lipid antioxidant throughout the cell and an electron transporter within the electron transport chain. isp-1 encodes an iron-sulfur component of complex III in the ETC. As both mutations affect respiration and display impaired development [22], [23], we hypothesized that they would cause activation of the UPR^mt. Indeed, hsp-60_pr::gfp expression was consistently elevated in both strains consistent with the presence of mitochondrial stress. The isp-1(qm150) mutation caused considerably stronger hsp-60_pr::gfp induction suggestive of a larger impact on mitochondrial function [29] (Figure 1B). Chaperone induction in both mutants required ATFS-1 as animals raised on atfs-1(RNAi) were unable to induce expression of hsp-60_pr::gfp (data not shown).

To determine if the ATFS-1-dependent regulation of mitochondrial chaperone genes has a protective role during mitochondrial stress we examined the effect of atfs-1(RNAi) on the development of wild-type and mitochondrial stressed worms. As previously demonstrated, both clk-1(qm30) and isp-1(qm150) worms developed considerably slower than wild-type animals [22], [28]. Consistent with ATFS-1 being a stress responsive transcription factor, wild-type worms fed atfs-1(RNAi) developed at similar rates to wild-type animals (data not shown). However, feeding clk-1(qm30) and isp-1(qm150) worms atfs-1(RNAi) dramatically impaired their developmental rates (Figure 1C and Figure S1), indicating a requirement for ATFS-1 during development in the presence of mitochondrial stress.

An RNAi Screen Suggests a Role for Translational Regulation in Protecting against Mitochondrial Stress

In addition to ATFS-1-regulated mitochondrial chaperone expression, we sought to identify additional components that promote mitochondrial protein homeostasis by acting in complementary pathways. To identify signaling pathways that act in parallel to ATFS-1 we generated and screened an RNAi sub-library consisting of all C. elegans kinases and phosphatases [30]. We took advantage of hsp-60_pr::gfp activation as a sensitive readout for the status of mitochondrial function to identify signaling components that promoted or impaired mitochondrial protein homeostasis. The clk-1(qm30) strain was chosen for the RNAi screen as it displayed mild hsp-60_pr::gfp induction, potentially allowing for the identification of candidates whose knockdown by RNAi either decreased or further increased hsp-60_pr::gfp expression (Figure 1B). We hypothesized that RNAi knockdown of candidates that act in a complementary protective signaling pathway would show enhanced hsp-60_pr::gfp activation in the presence of stress because of an increased substrate load on the mitochondrial protein folding machinery. Alternatively, if components exist whose knockdown somehow enhances the protein-folding capacity, then those RNAi may suppress hsp-60_pr::gfp activation in the clk-1(qm30) background.

Interestingly, RNAi of several kinases required for protein synthesis reduced hsp-60_pr::gfp expression in the clk-1(qm30) background (Figure 2A), which was also confirmed using the isp-1(qm150) strain (Figure S2A), suggesting that knockdown of these components protected the mitochondrial folding environment. The reduced hsp-60_pr::gfp expression was not simply due to a reduction in translation as separate GFP reporters under the myo-3, ges-1 or ER stress-inducible hsp-4 promoters were unaffected by the RNAi candidates (data not shown). These findings are consistent with previous experiments demonstrating that translation attenuation is protective against mitochondrial stress in yeast and Drosophila [12], [13]. Similarly, reduced translation has been associated with longevity and stress resistance in C. elegans. For example, knockdown of the C. elegans target of rapamycin ortholog (TOR), CeTor, which regulates mRNA translation in response to nutrient cues [31], or knockdown of rsks-1, the ribosomal S6 kinase, slows development and extends lifespan in C. elegans [16]. Because the long-lived clk-1(qm30) mitochondrial mutants have increased levels of mitochondrial stress and the stress responsive hsp-60_pr::gfp reporter was specifically reduced by CeTor and rsks-1(RNAi), these findings support the hypothesis that reduced translation is beneficial to mitochondrial protein homeostasis. However, because the TOR-signaling pathway impacts many biological processes in addition to translation, other possibilities exist. Because all components identified in our RNAi screen affect protein synthesis, we sought to further characterize the role of translation attenuation in maintaining the mitochondrial protein-folding environment.

GCN-2 Phosphorylates eIF2α in Response to Mitochondrial Stress

In addition to CeTor, rsks-1 and cel-1, we identified components that are known to regulate translation initiation by modulating the phosphorylation status of the translation initiation factor eIF2α. RNAi-knockdown of the eIF2α kinase General Control Non-derepressible-2 (GCN-2) further increased hsp-60_pr::gfp expression in clk-1(qm30) animals, suggesting a role for GCN-2 in promoting mitochondrial protein homeostasis or function (Figure 2A and 2B). The effect of gcn-2(RNAi) on hsp-60_pr::gfp expression was not due to direct effects on GFP translation as gcn-2(RNAi) did not cause induction of the ER stress reporter hsp-4_pr::gfp (Figure 3A) suggesting a specific role for GCN-2 in promoting mitochondrial protein homeostasis. In unstressed animals, gcn-2(RNAi) did not effect hsp-60_pr::gfp expression, suggesting its primary role is during stress (Figure 2B). Contrary to gcn-2(RNAi), our RNAi screen identified gsp-1(RNAi), which reduced hsp-60_pr::gfp expression in both the clk-1(qm30) and isp-1(qm150) strains (Figure 2A, 2B and Figure S2B). GSP-1 encodes a protein phosphatase (PP1) required for numerous cellular dephosphorylation events [32], [33] and is homologous to the yeast phosphatase required for eIF2α dephosphorylation [34].

To determine if GCN-2 and GSP-1 regulate eIF2α phosphorylation in C. elegans, we examined the phosphorylation status of eIF2α in whole worm lysates. We utilized an antibody that specifically recognizes the highly conserved serine that is phosphorylated by the repertoire of eIF2α kinases (S51 in mammals and S49 in C. elegans (Figure 3B)). Consistent with previous reports, we detected phosphorylated eIF2α in otherwise unstressed worms (Figure 3C and 3D) [35], [36], [37], which was reduced when the lysate was incubated with calf-intestine phosphatase (CIP), confirming the specificity of the antibody for the phosphorylated form of eIF2α (Figure 3C). Furthermore, in a deletion mutant lacking 1482 bases of gcn-2 (gcn-2(ok871)), the level of steady-state phospho-eIF2α was reduced relative to wild-type worms (Figure 3D). In C. elegans, the only other known eIF2α kinase is PEK-1 (homologous to mammalian PERK [38]). Indeed, phospho-eIF2α was further reduced relative to levels of total eIF2α protein and mRNA in a strain lacking both kinases (Figure 3D and Figure S3B), further supporting the specificity of the phospho-eIF2α antibody and demonstrating the contribution of both kinases to steady state levels of eIF2α phosphorylation.

In contrast to inhibition of GCN-2 and PEK-1, GSP-1 knockdown resulted in increased levels of phospho-eIF2α consistent with it acting as a constitutive eIF2α phosphatase (Figure 3E). In either the gcn-2(ok871) or pek-1(zcdf2) deletion strains fed gsp-1(RNAi) there was still an increase in steady state levels of eIF2α phosphorylation likely reflecting the ability of both kinases to constitutively phosphorylate eIF2α in the absence of exogenous stress (Figure 3E). As increased phospho-eIF2α results in reduced cytosolic translation [5], [39], these data suggest that gsp-1(RNAi) reduces hsp-60_pr::gfp induction through attenuation of cytosolic translation, thus reducing the load on the mitochondrial protein folding machinery similar to eIF2α phosphorylation and translation attenuation in the UPR^ER [5], [6].

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-60_pr::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.

It should be noted that deletion or knockdown of the other C. elegans eIF2α kinase PEK-1 had no obvious effect on hsp-60_pr::gfp induction during mitochondrial stress (data not shown). Furthermore, the increase in eIF2α phosphorylation observed in the clk-1(qm30) animals was not dependent on pek-1 indicating GCN-2 is the primary eIF2α kinase involved in maintaining mitochondrial protein homeostasis (Figure 4C). In pek-1 deletion worms, steady state levels of phospho-eIF2α were reduced (Figure 3E), however these animals still induce eIF2α phosphorylation in response to mitochondrial dysfunction supporting the specific role for GCN-2 during mitochondrial stress (Figure 4C). A similar relationship has been described with PEK-1 and the induction of ER chaperones during ER stress. PEK-1 is specifically activated during ER stress and animals lacking PEK-1 display stronger induction of ER chaperone genes including hsp-4 during ER stress [38]. The UPR^ER reporter hsp-4_pr::gfp is induced during mild heat stress, a condition known to activate the UPR^ER but not the UPR^mt [9], [11]. Incubation of hsp-4_pr::gfp animals at 30°C for 3 hours mildly induced GFP expression (Figure 3A and Figure S3A). However, worms raised on pek-1(RNAi) displayed a much stronger induction of the UPR^ER reporter upon heat exposure consistent with PEK-1 activity protecting ER protein homeostasis [19], [38]. Unlike pek-1(RNAi), gcn-2(RNAi) had no impact on hsp-4_pr::gfp during heat stress. These results indicate that the effect of gcn-2(RNAi) on hsp-60_pr::gfp induction was not due to dysregulation of global translation further supporting a mitochondrial stress-specific role for GCN-2.

Loss of GCN-2 Sensitizes Worms to Conditions That Induce Mitochondrial Stress

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.

To further assess the role of GCN-2 in maintaining mitochondrial function during mitochondrial stress we examined the effect of gcn-2 deletion on oxygen consumption in wild-type as well as mitochondrial stressed worms. We observed no difference in the rates of oxygen consumption between wild-type worms and those lacking gcn-2 (Figure 5D), consistent with the gcn-2(ok871) deletion having no effect on worm development (Figure S4). clk-1(qm30) worms displayed a slight reduction in oxygen consumption when compared to wild-type worms consistent with mild mitochondrial dysfunction (Figure 5E) [40]. Impressively, clk-1(qm30) worms lacking gcn-2 had a much lower rate of oxygen consumption than worms harboring either the gcn-2-deletion or clk-1(qm30) alone (Figure 5E), supporting a role for GCN-2 in promoting mitochondrial function during mitochondrial stress.

Elevated ROS produced by dysfunctional mitochondria can damage proteins through the formation of irreversible carobonyl modifications on lysine, cysteine, proline and threonine residues [41], [42], [43]. In order to examine levels of oxidative damage in mitochondrial stressed worms, we visualized the accumulation of carbonylated proteins using the Oxyblot system [44]. Consistent with the clk-1(qm30) mutation causing mitochondrial dysfunction, significantly more carbonylated material was detected in lysates from clk-1(qm30) worms than lysates from wild-type worms (Figure 5F). clk-1(qm30);gcn-2(ok871) worms displayed even more oxidative damage than worms harboring clk-1(qm30) alone. Because oxidative damage can perturb protein folding, these data support a role for GCN-2 in protecting the folding environment as well as mitochondrial function.

To further assess the contribution of GCN-2 in maintaining mitochondrial protein homeostasis we targeted GFP to the mitochondrial matrix via the strong muscle-specific myosin promoter (myo-3). High-level expression of mitochondria-targeted GFP challenges the organelle's protein folding environment by increasing the load of unfolded proteins [24], [45]. While wild-type worms were able to accommodate the increased folding load and maintain mitochondrial morphology, myo-3_pr::gfp^mt worms raised on gcn-2(RNAi) displayed severely perturbed mitochondrial morphology consistent with a loss of protein homeostasis and mitochondrial function [24], [46] (Figure 5G). Furthermore, in the absence of GCN-2, developmental rates (data not shown) and muscle cell function were severely reduced as determined by a motility or thrashing assay (Figure 5H). Together these data indicate that GCN-2 protects mitochondrial function during increased load of mitochondrial unfolded proteins.

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

clk-1(qm30) and isp-1(qm150) animals, which activate ATFS-1-dependent hsp-60_pr::gfp expression and GCN-2-dependent eIF2α phosphorylation, are among the numerous C. elegans mitochondrial mutants that exhibit lifespan extension [47], [48], [49]. It was recently reported that ubl-5, a small ubiquitin-like protein required for UPR^mt signaling [45], was required for lifespan extension in several mitochondrial mutants highlighting the importance of maintaining mitochondrial protein homeostasis [50]. Consistent with these studies, knockdown of ubl-5 prevented hsp-60_pr::gfp induction in the long-lived clk-1(qm30) worms (Figure S5) similar to atfs-1(RNAi).

Additionally, cytosolic translation attenuation also contributes to longevity in several animal models [15], [16], [17]. As GCN-2 slows cytosolic translation [51] in response to mitochondrial dysfunction, we examined the role of GCN-2 in lifespan extension associated with mitochondrial dysfunction. Interestingly, GCN-2 knockdown in clk-1(qm30) animals reduced their lifespan to that of wild-type worms (Figure 6A) consistent with a role for GCN-2 in lifespan extension associated with mitochondrial dysfunction. gcn-2(RNAi) was not generally toxic, as it did not affect lifespan or development in the absence of stress (Figure 6B and Figure S4). gcn-2(RNAi) also shortened the lifespan of isp-1(qm150) animals, but because the animals were very sick with considerable developmental defects, we were unable to determine a role for GCN-2 in longevity of these animals (data not shown). These data are consistent with GCN-2 and increased eIF2α phosphorylation contributing to the lifespan extension observed in mitochondrial mutants and further emphasizes the importance of protein homeostasis in aging.

Because gsp-1(RNAi) caused an increase in eIF2α phosphorylation in the absence or presence of mitochondrial stress (Figure 3E, Figure 4A and 4B), we hypothesized gsp-1(RNAi) would promote lifespan extension. However, the lifespan of wild-type or clk-1(qm30) worms on gsp-1(RNAi) were severely shortened (Figure S6). Interpretation of this result is complicated by the pleitropic, non-specific effects of GSP-1 knockdown. gsp-1(RNAi) also prevents C. elegans germline formation (data not shown) and is required for a variety of cellular processes including mitosis [33]. Therefore, we were unable to determine if increased eIF2α phosphorylation was sufficient to extend lifespan.

ROS Are Required for GCN-2–Dependent eIF2α Phosphorylation during Mitochondrial Stress

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-60_pr::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-60_pr::gfp activation, similar to gcn-2(RNAi) (Figure 7A and Figure 2B). Ascorbate had no effect on the induction of hsp-60_pr::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).

Our data indicate a requirement for ROS in GCN-2-dependent eIF2α phosphorylation observed in response to mitochondrial dysfunction in clk-1(qm30) and isp-1(qm150) mutants. In addition to these two mutants which generate ROS, the herbicide paraquat is known to generate excessive ROS and extend C. elegans lifespan [22], [23]. Interestingly, similar to the clk-1(qm30) and isp-1(qm150) mutants, exposure of wild-type worms to paraquat increased eIF2α phosphorylation in a GCN-2-dependent manner (Figure 7C). In sum, these data support a protective upstream signaling role for mitochondria-generated ROS in GCN-2 activation during mitochondrial stress.

GCN-2 Acts in a Complementary Pathway to That of ATFS-1 and Mitochondrial Chaperone Induction

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-60_pr::gfp expression. Similarly, reducing eIF2α phosphorylation by inhibiting ROS accumulation resulted in increased activation of hsp-60_pr::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.

We next investigated the potential synthetic interaction between GCN-2 and ATFS-1 during worm development. Either individual mutation had no obvious growth or developmental defect in the absence of stress (Figure 8B and Figure S4). However, gcn-2(ok871);atfs-1(tm4525) animals or the gcn-2(ok871) strain fed atfs-1(RNAi) developed somewhat slower in the absence of exogenous stress (Figure 8B and 8C). These data suggest the presence of low levels of mitochondrial stress during development that required the activity of either GCN-2 or ATFS-1.

In the presence of stress caused by spg-7(RNAi) or the clk-1(qm30) mutation, development of worms lacking gcn-2 and atfs-1 was severely compromised. When raised on spg-7(RNAi), most worms arrested at the L1 or L2 larval stage and no animals were able to reach adulthood (Figure 8C and Figure S7A). Furthermore, clk-1(qm30) (Figure 8D and Figure S7B) or isp-1(qm150) (data not shown) animals lacking both GCN-2 and ATFS-1 developed more slowly than worms lacking either individual gene. No synthetic interactions were observed between ATFS-1 and PEK-1 as atfs-1(tm4525) animals raised on pek-1(RNAi) developed at similar rates to atfs-1(tm4525) animals raised on a control RNAi in the absence or presence of stress (data not shown). Despite the developmental defect observed in animals lacking both ATFS-1 and GCN-2 (Figure 8B and 8C), animals lacking both genes had similar lifespans to those of wild-type worms (Figure 8E) suggesting the primary role for each pathway in the absence of exogenous stress is during development, when the majority of mitochondrial biogenesis occurs [54]. Together, these results support a model in which ATFS-1 and GCN-2 act in different yet complementary mitochondrial stress response pathways to regulate mitochondrial chaperone expression and cytosolic translation to protect mitochondrial function (Figure 8F).

C. elegans Strains and Growth Conditions

Reporter strains hsp-60_pr::gfp(zcIs9)V, myo-3_pr::gfp^mt(zcIs14) and hsp-4_pr::gfp(zcIs4)V have been described previously [9], [24], [45]. Where indicated, the hsp-60_pr::gfp(zcIs9)V transgene was crossed into individual mutant strains of interest, with the exception of atfs-1(tm4525)V, which was backcrossed with N2 three times prior to crossing into the hsp-60_pr::gfp background. The clk-1(qm30), isp-1(qm150) and gcn-2(ok871) strains were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN) and the atfs-1(tm4525) strain was obtained from the National BioResource Project (Tokyo, Japan). RNAi feeding experiments were performed as described [9] with constructs from the Ahringer and Vidal libraries [66], [67].

Development and Lifespan Analysis

Worms were synchronized via bleaching and allowed to develop on the described RNAi plate or condition. For development in the presence of oxidative stress, rotenone was applied to vector(RNAi) plates and allowed to soak in prior to seeding eggs. At the time points indicated, the numbers of L1, L2, L3, L4, young adult (non-gravid) or gravid adult worms were counted on each plate and quantified as a percent of the total number of animals. For each plate, the worms in 6–8 individual fields of view were counted, and the total number combined.

For lifespan analysis, worms were synchronized as eggs and allowed to develop under the described condition for two days. At that point, 100 L4 animals were transferred to new RNAi plates and subsequently transferred to fresh plates every day for the next 5–6 days and every two days thereafter. The numbers of dead and censored worms were counted every second day for the duration of the assay [68]. Survival curves and statistical analysis were generated using Prism 5.0b software (Graphpad). Each experiment was repeated 3 times.

Western Blots

Worms were grown under the described conditions and collected at the L4 stage for analysis as previously described [9], [24]. Phospho-eIF2α antibody (#3597S) was obtained from Cell Signaling Technology (Danvers, MA) and observed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockville, IL). GFP and HDEL immunoblots were visualized using Odyssey Infrared Imager (Li-Cor Biosciences, Lincoln, NE). Total eIF2α was assessed as described [36]. Due to limited amounts of pan-eIF2αantibody, western analysis was only performed during select experiments to confirm specificity of the phospho-eIF2α antibody in wild-type lysates and during analysis phospho-eIF2α levels in clk-1(qm30) animals.

For the eIF2α dephosphorylation assay, 150 µg of worm lysate was treated with calf intestinal phosphatase (CIP) for 30 minutes at 30°C prior to SDS-PAGE analysis. For ascorbate treatment, synchronized worms were grown to adulthood in liquid medium, when 25 mM ascorbate was added for 16 hours prior to western analysis.

RNA Isolation, cDNA Synthesis, and Quantitative RT–PCR

Total RNA was isolated using RNA STAT (Tel-Test Inc, Friendswood, TX). RNA samples were prepared from the described worms at the L4 stage. cDNA was then synthesized from total RNA using a iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Following mRNA isolation and cDNA synthesis, qPCR was used to determine the expression level of eif2α using iQ sybr green supermix and MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad). Actin was used as a control. Fold changes in gene expression were calculated using the comparative CtΔΔCt method.

Microscopy

Fluorescent photomicrographs were obtained using a Zeiss AxioCam MRm mounted on a Zeiss Imager.Z2 microscope or Zeiss M2 Bio stereo microscope (Carl Zeiss Imaging, Thornwood, NY).

Oxygen Consumption and Examination of Oxidative Protein Modification

Oxygen consumption assays were performed as described [11] using a Clark type electrode [40]. To determine the accumulation of oxidative protein modifications, synchronized wild-type, clk-1(qm30) and clk-1(qm30);gcn-2(ok871) worms were harvested once they reached the L4 stage. Worm lysates were separated by SDS-Page and treated according to the Oxyblot manufacturer (Millipore, Billerica, MA).