Mitochondrial SKN-1/Nrf mediates a conserved starvation response

Abstract

SKN-1/Nrf plays multiple essential roles in development and cellular homeostasis. We demonstrate that SKN-1 executes a specific and appropriate transcriptional response to changes in available nutrients, leading to metabolic adaptation. We isolated gain-of-function (gf) alleles of skn-1, affecting a domain of SKN-1 that binds the transcription factor MXL-3 and the mitochondrial outer membrane protein PGAM-5. These skn-1(gf) mutants perceive a state of starvation even in the presence of plentiful food. The aberrant monitoring of cellular nutritional status leads to an altered survival response in which skn-1(gf) mutants transcriptionally activate genes associated with metabolism, adaptation to starvation, aging, and survival. The triggered starvation response is conserved in mice with constitutively activated Nrf and may contribute to the tumorgenicity associated with activating Nrf mutations in mammalian somatic cells. Our findings delineate an evolutionarily conserved metabolic axis of SKN-1/Nrf, further establishing the complexity of this pathway.

Figure 1. The Dominant SKN-1 Domain Functions Independently of the Canonical WDR-23 Pathway

(A) SNP mapping with the Hawaiian wild-type strain CB4856 assigned alleles lax120 and lax188 to LGIV between B0273 and C49H3. 576 RNAi clones covering the genes between these SNPs were tested for suppression of the dominant phenotype. (B) A skn-1/T19E7.2 RNAi clone was the only construct capable of suppression of the dominant phenotype. (C) lax120 and lax188 encode single missense mutations in the skn-1 coding sequence. Y2H analysis was performed with bait constructs that code for a 200, 100, or 50 amino acid region mutated in the dominant skn-1 alleles. (D) skn-1(lax120) and skn-1(lax188) mutations alter the interaction of the SKN-1 50 amino acid domain with Y2H targets.

Figure 2. Identification of a Unique Mitochondria-Associated SKN-1 Activation Pathway

(A) SKN-1 is enriched in a mitochondria fraction of whole C. elegans. The pL4440 and skn-1 RNAi controls are from whole-worm extracts. In the left panel, asterisks denote bands that are not clearly decreased by skn-1 RNAi, in contrast to 85 kD SKN-1 itself. (B) Partial purification was achieved by spinning down the mito pellet (mito), as indicated by the presence of remaining mitochondrial marker, PDHE1 (Greiss et al., 2008; Li et al., 2011; Brys et al., 2010), in the cytosolic fraction (cyto), but the mitochondria fraction lacks the cytoplasmic marker GAPDH. (C) Arrowhead indicates remaining PDHE1 that was not removed by stripping. (D) Synergistic activation of the SKN-1 transcriptional reporter (gst-4p::gfp) by the canonical wdr-23 pathway and the dominant skn-1 mutations. Percentage represents fraction of total animals, n, with phenotype. (E) GFP score of indicated genotypes based on fluorescence intensity.

Figure 3. SKN-1 Is Activated by Starvation and Changes in Available Nutrients

(A) GO-terms of genes identified as dysregulated by transcriptional analysis of the lax120 and lax188 mutants. (B) SKN-1 activity sensor worms were grown on food or under starvation conditions and then imaged for GFP expression indicative of SKN-1 activation of the gst-4p::gfp reporter. (C) Quantification of GFP levels observed as described in (B). (D–I) Indicated strains were fed the E. coli B strain OP50. (D)–(F) or the E.coli K12 strain HT115 (G)–(I) and imaged for GFP expression as described in (B).

Figure 4. Activated Mito-SKN-1 Promotes a Perceived State of Starvation

(A) Bacterial dilution (bDR) life span; the mean life span of adult worms grown on solid media with four concentration of bacteria at 25°C. (B) Feeding rates as assessed by pharyngeal pumping assays for indicated strains. Error bars represent average +SD.

Figure 5. Activated SKN-1 Promotes Extended Reproduction and Developmental Delay in Response to Food

(A) Nonmated reproductive span of worms fed OP50 bacteria at 20°C. (B) Total progeny from (A). (C) L1 diapause survival was measured by movement and staining with bromophenol blue following 5, 7, and 12 days in the absence of food. (D) Growth and development of animals in L1 diapause following feeding. (E) Percentage of animals arrested early in development after recovery from starvation. (* < 0.05, ** < 0.01, *** < 0.001). Error bars represent SEM.