Activation of Target of Rapamycin Complex 1 S/T kinase (TORC1) by the Rheb small GTPase is a major signaling axis that is conserved from yeasts to humans. TORC1 integrates inputs from both local and systemic nutrients to promote anabolism and growth. Activated TOR complex 1 (TORC1; defined by the presence of DAF-15/Raptor) promotes biosynthesis of proteins and other macromolecules while inhibiting autophagy/catabolism. TORC1 signaling has been mainly studied using in vitro cell culture, and Rheb itself has received relatively little attention. Here, we use C. elegans as a model to dissect the Rheb-TORC1 signaling axis in vivo. A few C. elegans genetic reagents for the Rheb-TOR axis have been defined: null or strong loss-of-function mutations in
let-363/TOR and
daf-15/Raptor cause developmental arrest at the 3rd larval stage. We found that deletion alleles of
rheb-1 conferred arrest at a size much smaller than the L3 arrest described for disruption of Raptor or TOR. We interpret the Rheb mutant arrest to be at the L2 stage, and we confirmed this result by assaying the HLH-8::GFP M-linage reporter in the
rheb-1 mutant. We hypothesize that DAF-15/Raptor and LET-363/TOR mutations are maternally rescued, and preliminary depletion of maternal DAF-15/Raptor led to a range of arrest phenotypes, including putative L2. Arrested
rheb-1 mutants move and pump, and they survive for a week after arrest. Mutation of DAF-16/FOXO significantly decreases
rheb-1 mutant arrest lifespan, while mutation of InsR/DAF-2 extends the
rheb-1 mutant lifespan almost three fold. Neither
daf-16 nor
daf-2 mutations alter stage of
rheb-1 mutant arrest. These results suggest that Insulin/IGF signalling (IIS) functions in parallel to TORC1. We hypothesize that the
rheb-1 null phenotype is a diapause/arrest rather than lethality, suggesting a TORC1 requirement in developmental progression. To avoid perturbing membrane-targeting sequences at the Rheb C-terminus, we used CRISPR to N-terminally tag endogenous Rheb, and observed expression in all tissues, with strong localization to lysosomes and modest localization to plasma membranes. RHEB-1 co-localizes with lysosome markers such as LMP-1::GFP and GFP::RAB-7, but not with GFP::RAB-5 (a marker of early endosomes). We also used CRISPR to C-terminally tag the endogenous DAF-15/Raptor. We observed Raptor in all tissues, localized to the cytosol and lysosomes. We will use co-localization of Rheb and Raptor to investigate Rheb-dependent recruitment of Raptor to the lysosome, comparing between well-fed and dietary restricted animals. Further, we also developed a putative constitutively activated TORC1 by knock-in of membrane targeting sequence of RHEB-1 into the DAF-15/Raptor C-terminus, and we hypothesize that this protein fusion might be able to rescue the arrest phenotype of the
rheb-1 mutant.