Rheb small GTPase activation of Target of Rapamycin (TOR) S/T kinase is a major signaling axis controlling anabolic metabolism that is conserved from yeasts to humans. TOR integrates inputs from both local and systemic nutrients to orchestrate growth. Activated TOR complex 1 (TORC1; defined by the presence of Raptor/DAF-15) induces protein synthesis, builds macromolecules and inhibits autophagy. A few C. elegans genetic reagents for the Rheb-TOR axis have been defined: null alleles of
let-363/TOR and
daf-15/Raptor cause L3 developmental arrest. We characterized outcrossed
rheb-1 null deletion alleles
tm4931 and
tm4642 and found that they confer L2 larval arrest. This strong loss-of-function arrest phenotype makes it difficult to study genetic interactions with other pathways, particularly their functions at later points in life, such as growth rate and aging. Furthermore, Rheb-TORC1 signaling has been studied mainly using in vitro cell culture, and Rheb itself has received relatively little attention. We are studying the Rheb-TORC1 signaling axis using two main approaches. First, we are investigating
rheb-1 genetically and cell biologically. Unusually for a small GTPase, whose functions are generally tightly regulated by GEFs and GAPs, RHEB-1 over-expression is toxic, suggesting a possible mechanism of TOR regulation through controlling RHEB-1 expression. We have engineered a conditional RHEB-1 expression system using a heterologous promoter with a nonsense mediated mRNA decay (NMD)-sensitive 3'UTR and the
smg-1(ts) allele. We are also using CRISPR to tag the endogenous
rheb-1 locus to track both expression and subcellular localization. Second, we are creating a genetic toolkit for Rheb-TORC1 analysis by assessing viable
let-363,
rheb-1 and
daf-15 point mutants generated by the Million Mutation Project (MMP). We selected 22 (out of 51) viable
let-363 MMP candidates; this preliminary selection was based on mutation sites at conserved residues and in critical domains that are likely to confer reduced or gain of TOR function. We tracked developmental rate of these
let-363 MMP mutant animals grown on Raptor/daf-15(RNAi) to sensitize the background. Wild types were mildly growth delayed while our best candidates exhibited substantial growth retardation. We identified four potential TOR reduced function alleles. We will further confirm these alleles using different genetic backgrounds, and then use them as tools for interrogating novel components of the Rheb-TORC1 system. We will evaluate their impact on metabolism, fat storage and aging. Taken together, establishing a Rheb-TORC1 experimental system will provide powerful means to investigate the mechanisms of TORC1 control of metabolism and aging in vivo.