Conserved Pib2 regions have distinct roles in TORC1 regulation at the vacuole.

TORC1 is a critical controller of cell growth in eukaryotes. In yeast (Saccharomyces cerevisiae), the presence of nutrients is signaled to TORC1 by several upstream regulatory sensors that together coordinate TORC1 activity. TORC1 localizes to both vacuolar and endosomal membranes, where differential signaling occurs. This localization is mimicked by Pib2, a key upstream TORC1 regulator that is essential for TORC1 reactivation after nutrient starvation or pharmacological inhibition. Pib2 has both positive and negative effects on TORC1 activity, but the mechanisms remain poorly understood. Here, we pinpoint the Pib2 inhibitory function on TORC1 to residues within short, conserved N-terminal regions. We also show that the Pib2 C-terminal regions, helical region E and tail, are essential for TORC1 reactivation. Furthermore, the Pib2 FYVE domain plays a role in vacuolar localization, but it is surprisingly unnecessary for recovery from rapamycin exposure. Using chimeric Pib2 targeting constructs, we show that endosomal localization is not necessary for TORC1 reactivation and cell growth after rapamycin treatment. Thus, a comprehensive molecular dissection of Pib2 demonstrates that each of its conserved regions differentially contribute to Pib2-mediated regulation of TORC1 activity.