Figure 1. SGK-1 affects SMF-1 protein- and iron levels to control lifespan:A-B: FerroOrange staining and its quantification. Adult day 5
sgk-1(lf) mutants accumulate iron in the anterior and posterior intestine compared to wild type (A). 10-15 worms of each genotype were used for quantification (B). Two-tailed t-test, mean ± SD; results are representative for two biological replicates. C: RNAi-mediated knock-down of
mfn-1 (mitoferrin 1) from hatching increases lifespan in
sgk-1(lf) mutants. n ≥ 100. Results are representative for two biological replicates. D-E: Bacterial avoidance assay.
sgk-1(lf) mutants show higher bacterial avoidance from adult day 1 to day 6 compared to wild type (D), which is suppressed upon treatment with 50 µM iron chelator (phe) (E). P values were calculated using a two-tailed t-test for each time point, mean ± SD. Results are representative for two biological replicates. F-H: Adult day 1
sgk-1(lf) mutants accumulate SMF-1::GFP in the anterior and posterior intestine. Images are taken using a 20X lens (F) and a 63X lens (H). Arrows in H indicate the apical membrane localization of SMF-1::GFP. Quantification of SMF-1::GFP fluorescence (G) from images in (F) was performed using 10-15 worms of each genotype. Two-tailed t-test for each time point, mean ± SD. Results are representative for three biological replicates. I: RNAi-mediated knock-down of
smf-1, encoding a divalent cation transporter, increases lifespan in
sgk-1(lf) mutants. n ≥ 73. Results are representative for two biological replicates. The
sgk-1(
ok538) loss-of-function mutant was used for all experiments (A-I). *P-value <0.05, **P-value <0.01, ***P-value <0.001 for two-tailed t-test (A-I). J: Working model. SGK-1 expression is induced upon iron overload, which reduces free iron by promoting FTN-1 expression and reducing SMF-1 levels likely through facilitating SMF-1's recycling through endocytosis. When SGK-1 function is impaired, SMF-1 might accumulate on intestinal cell membranes resulting in elevated iron uptake. Iron overload induces ROS production and mitochondrial dysfunction, which is detrimental to animals' survival. Mitophagy might further accelerate ROS production through Fenton's reaction by providing a low pH environment.