EGO-like complex regulates TOR (Target of Rapamycin) activity and localization in Neurospora

The TOR (Target of Rapamycin) signalling pathway is found in all eukaryotes and integrates nutrient and stress signals to control cell growth. It is well-studied in yeast and mammals but is less well understood in filamentous fungi. We previously identified TOR pathway components VTA (homologous to the vacuole-bound EGO complex of yeast) and GTR2 (homologous to Rag GTPases) as essential to maintaining circadian rhythms in the filamentous fungus Neurospora crassa. Therefore we are interested in defining the TOR pathway and its regulation in N. crassa. In yeast and mammals, TOR kinase is activated by carbon sources and amino acids. We report here that on high glucose medium, TOR is insensitive to added amino acids. On low glucose, TOR is activated by added glucose and amino acids. VTA and GTR2 knockouts block the activation of TOR by amino acids but not by glucose, identifying their function in an amino acid-sensing pathway. Live cell microscopy of KOG1 (a component of TOR complex 1) and GTR2 localizes them to punctate bodies near the vacuole. This localization is lost and the proteins are largely cytoplasmic under starvation conditions, in the presence of TOR inhibitor Torin II, and in the VTA knockout. This indicates that VTA acts as the vacuolar anchor for activated TOR complex. Co-immunoprecipitation of KOG1-FLAG and GTR2-FLAG confirms their cytoplasmic localization in VTA knockout and identifies TORC1 complex components TCO89 and LST8. These results focus attention on amino acid sensing through VTA and GTR2 as potentially regulating circadian rhythmicity in N. crassa.