<i>natc-1</i> mutations cause resistance to multiple metals.

<p>Wild-type (WT) and <i>natc-1(am138)</i> embryos were cultured on NAMM supplemented with (A) 200 µM zinc, (B) 20 µM cadmium, (C) 50 µM nickel, or (D) 300 µM copper. Bars display the percentage of fertile adults (N = 37–52). *, p<0.05 compared to WT.</p

NATC-1 protein is expressed in many cells and tissues and localizes to the cytoplasm.

<p>We generated transgenic <i>natc-1(am138)</i> animals expressing NATC-1::GFP fusion protein driven by the <i>natc-1</i> promoter (WU1449). Confocal fluorescent microscope images of live animals are shown with head on the left and tail on the right. Green represents NATC-1::GFP fusion proteins, except for puncta in intestinal cells visible in panels A, C and D that reflect autofluorescent gut granules. (A) An image of an entire worm. NATC-1::GFP signal is visible in many cells and tissues throughout the animal, and the uniform staining pattern suggests cytoplasmic localization. (B–E) Higher magnification images display fluorescence in the pharynx (P>), sheath cells (SC>), intestinal cells (I*), distal tip cell (DTC>), vulva (V*) and body wall muscle (BWM>). Scale bar is 25 µm for all images.</p

<i>natc-1</i> mutations increase resistance to heat and oxidative stress.

<p>(A) Wild-type (WT) and <i>natc-1(am138)</i> animals were cultured at 15°C on NGM until day 1 of adulthood, shifted to 35°C, and assayed for survival hourly, which involved a brief exposure to room temperature. (B) WT and <i>natc-1(am138)</i> animals were cultured at 15°C on NGM until day 3 of adulthood, shifted to NGM containing 40 mM paraquat to induce oxidative stress, and assayed for survival every 12 hours. (C) WT and <i>natc-1(am138)</i> animals were cultured at 20°C on NGM and assayed for survival every day. Day 0 is defined as the L4 stage of development (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen-1004703-t001" target="_blank">Table 1</a> for summary statistics).</p

<i>natc-1</i> is a physiologically significant downstream effector of the insulin/IGF-1 signaling pathway.

<p>(A) A genetic model of <i>natc-1</i> function. Arrows and bars indicate positive and negative interactions, respectively, that may be direct or indirect. We propose that <i>natc-1</i> negatively regulates stress resistance and dauer formation based on the phenotype of <i>natc-1(lf)</i> mutant animals and <i>natc-1</i> functions downstream of <i>daf-16</i> based on genetic epistasis results and regulation of <i>natc-1</i> mRNA levels. Other interactions are based on published studies <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Hu1" target="_blank">[1]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Hunter1" target="_blank">[54]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Gottlieb1" target="_blank">[88]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Honda2" target="_blank">[89]</a>. In contrast to <i>sod-3</i> and other well-characterized DAF-16 target genes that are positively regulated and enhance stress resistance, <i>natc-1</i> is negatively regulated and inhibits stress resistance. (B) A combined genetic and molecular model of the function and regulation of <i>natc-1</i>. DAF-2 is an insulin/IGF-1 transmembrane receptor that functions through a signaling pathway (not shown) to inhibit the activity of the DAF-16 FOXO transcription factor <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Gems1" target="_blank">[90]</a>. We propose DAF-16 directly binds the <i>natc-1</i> promoter and represses transcription. <i>natc-1</i> transcription and translation promotes activity of the NatC complex, which is predicted to acetylate multiple proteins. However, the role of specific target proteins that modulate stress resistance and dauer larvae formation has not been established.</p

<i>natc-1</i> is regulated by <i>daf-16</i> and functions in dauer formation.

<p>(A) A model of the <i>natc-1</i> promoter and open reading frame showing DAF-16 protein binding an evolutionarily conserved DAF-16 binding site positioned 90 base pairs upstream of the <i>natc-1</i> start codon <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Lee1" target="_blank">[11]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Gerstein1" target="_blank">[41]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Riedel1" target="_blank">[42]</a>. (B) Wild-type, <i>daf-2(e1370)</i>, and <i>daf-16(mu86);daf-2(e1370)</i> animals were synchronized at the L1 stage of development and cultured for 2 days at 20°C on NGM. For each genotype, mRNA was extracted, analyzed by qRT-PCR, and <i>natc-1</i> mRNA levels were normalized to the control gene <i>rps-23</i>. Bars show mRNA fold-change values calculated by comparing <i>daf-2</i> and <i>daf-16;daf-2</i> to WT by the comparative C_T method (N = 4–6 biological replicates). Error bars indicate standard deviation. <i>natc-1</i> mRNA levels were significantly reduced in <i>daf-2(e1370)</i> animals compared to WT but were not significantly different from WT in <i>daf-16;daf-2</i> animals (*, p<0.05). The <i>mtl-1</i> gene was utilized as a positive control, since it is an established target of DAF-16 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Barsyte1" target="_blank">[35]</a>, and <i>mtl-1</i> mRNA levels were significantly increased in <i>daf-2(e1370)</i> compared to WT (p<0.05) and this effect was <i>daf-16</i> dependent (p<0.05). (C) <i>daf-2(e1370)</i>, <i>natc-1(am138)</i>, and <i>daf-2(e1370);natc-1(am138)</i> embryos were cultured for 4 days on NGM at the indicated temperature and scored as either dauer or non-dauer (N = 200–341). Values for <i>daf-2;natc-1</i> were significantly higher than <i>daf-2</i> at 17.5°C, 20°C, and 22.5°C (*, p<0.05). (D) <i>daf-2(e1370)</i> embryos were cultured on NGM at 20°C, fed <i>E. coli</i> expressing dsRNA targeting <i>natc-1</i>, <i>natc-2</i>, or an empty vector control, and scored as dauer or non-dauer after 4 days (N = 590–627). <i>natc-1</i> and <i>natc-2</i> RNAi significantly increased dauer formation compared to control (*, p<0.05).</p

<i>natc-1</i> encodes a subunit of the NatC protein acetylation complex.

<p>(A) The <i>natc-1</i> mRNA structure. Boxes represent exons, and shading indicates untranslated regions. Black dotted lines indicate introns, SL1 (splice leader 1) indicates the 5′ trans-spliced leader sequence, and AAA indicates the 3′ polyA tail. <i>am138</i> (red) and <i>ok2062</i> (green) are deletion mutations, and <i>am134</i> is a nonsense mutation at codon 691 (blue). (B) The predicted <i>C. elegans</i> NATC-1 protein is aligned with homologous proteins CG4065 from the insect <i>Drosophila melanogaster</i> (Dm) and NAA35 from the vertebrate <i>Homo sapiens</i> (Hs) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Ruan1" target="_blank">[45]</a>. Shaded amino acids are identical to <i>C. elegans</i> NATC-1. Amino acids deleted by <i>am138</i> (red) and <i>ok2062</i> (green) are shown above. The blue-outlined asterisk indicates the codon mutated in <i>am134</i>. (C) A model based on the observations that NATC-1 and <i>C. elegans</i> NATC-2 (<i>B0238.10</i>) are homologous to an auxiliary and catalytic subunit of the NatC complex, respectively. The NatC complex catalyzes the acetylation of translating proteins at the N-terminus utilizing acetyl-CoA as a substrate. Xaa following Met is typically isoleucine, leucine, tryptophan, or phenylalanine <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Polevoda1" target="_blank">[39]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Polevoda2" target="_blank">[40]</a>.</p

<i>natc-1</i> mutations cause resistance to high zinc toxicity.

<p>(A) Wild-type (WT) and <i>natc-1(am138)</i> embryos were cultured on NAMM medium with <i>E. coli</i> as a food source and supplemental zinc (µM). Data points indicate the percent of embryos that developed to adulthood and were fertile based on the generation of at least one progeny (N = 25–226 animals). <i>natc-1(am138)</i> animals displayed significant resistance compared to wild-type animals at 200 µM and higher concentrations of supplemental zinc (*, p<0.05). (B) Wild-type, <i>natc-1(am134)</i>, <i>natc-1(am138)</i>, and <i>natc-1(ok2062)</i> embryos were cultured on NAMM supplemented with 250 µM zinc (N = 45–55). All three <i>natc-1</i> mutant strains displayed significant resistance to zinc toxicity compared to wild type (*, p<0.05). (C) A genetic map of the center of <i>C. elegans</i> linkage group (LG) V. Loci defined by SNPs and mutations that cause visible phenotypes are named above, and positions in map units are shown below. <i>am134</i> and <i>am138</i> displayed tightest linkage to the SNP <i>pkP5513</i> compared to the other SNP markers <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Bruinsma1" target="_blank">[36]</a>. Three-factor mapping experiments positioned <i>am138</i> between <i>dpy-11</i> and <i>unc-42</i>, an interval of 325 kb <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703-Bruinsma1" target="_blank">[36]</a> that includes <i>natc-1</i> (<i>T23B12.4</i>). Transgenic <i>natc-1(am134)</i> animals containing extrachromosomal arrays with plasmid pJM5, which encodes wild-type NATC-1, or plasmid pJM6, which encodes NATC-1 with a 112 amino acid deletion from codon 33–144 resulting in a predicted frameshift in the mutated open reading frame, were assayed for zinc resistance. Transgenic <i>natc-1(am138)</i> animals containing extrachromosomal arrays with plasmid pJM8, which encodes wild-type NATC-1 fused to green fluorescent protein (GFP), were assayed for zinc resistance. Open boxes indicate exons, and shading indicates untranslated regions. The GFP open reading frame is shaded in green. Rescue indicates the number of independently derived transgenic strains in which transgenic siblings displayed significantly reduced resistance to high zinc compared to nontransgenic siblings and the total number of transgenic strains analyzed (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen.1004703.s001" target="_blank">Figure S1</a></b>).</p

<i>natc-1</i> is epistatic to <i>daf-16</i> in resistance to heat and zinc stress.

<p>(A) Wild-type (WT), <i>natc-1(am138)</i>, <i>daf-2(e1370)</i>, and <i>daf-2(e1370);natc-1(am138)</i> animals were cultured at 15°C on NGM, shifted to 35°C as day 1 adults, and assayed for survival hourly beginning at 12 hours (N = 39–61). (B) Wild-type (WT), <i>natc-1(am138)</i>, <i>daf-16(mu86)</i>, and <i>daf-16(mu86);natc-1(am138)</i> animals were cultured at 15°C on NGM, shifted to 35°C as day 1 adults, and assayed for survival hourly. Summary statistics are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004703#pgen-1004703-t001" target="_blank">Table 1</a>. (C) Embryos were cultured on NAMM with 200 µM supplemental zinc. Bars indicate the percentage of embryos that generated fertile adults. Genotypes were wild type (WT), <i>natc-1(am138)</i>, <i>daf-16(mu86)</i>, and <i>daf-16(mu86);natc-1(am138)</i> (N = 49–54). <i>daf-16(mu86)</i> animals were similar to wild-type animals, and <i>natc-1(am138)</i> caused significant zinc resistance in wild-type and <i>daf-16(mu86)</i> mutant animals (*, p<0.05).</p