Proteotoxic stress, arising from conditions that cause misfolded protein accumulation, is closely linked to the pathogenesis of multiple diseases. Macroautophagy/autophagy activation is considered a compensatory mechanism to maintain protein homeostasis, but the underlying regulatory mechanisms remain incompletely understood. Here, we show that proteotoxic stress induced by proteasome inhibition, puromycin treatment, or polyglutamine-expanded HTT (huntingtin) expression promotes nuclear accumulation of TFEB and TFE3, key regulators of lysosomal biogenesis and autophagy. Mechanistically, TFEB activation under proteotoxic stress occurs independently of canonical MTORC1 inactivation mediated by TSC2 or ATF4. Instead, it involves non-canonical inhibition of MTORC1 via RRAG GTPases. Proteotoxic stress disrupts the RRAGC-TFEB interaction, preventing TFEB recruitment to lysosomes and subsequent MTORC1 phosphorylation. An activated RRAGC mutant rescues impaired lysosomal localization and nuclear accumulation of TFEB, while co-overexpression of FLCN and FNIP2, a GAP for RRAGC, partially restores stress-induced TFEB dephosphorylation. In addition, proteasome inhibition activates non-canonical autophagy. Deletion ofor, which known blocks Atg8-family protein lipidation and sequesters the FLCN-FNIP2 complex, partially abolishes proteotoxic stress-induced TFEB dephosphorylation and nuclear accumulation. Together, these findings demonstrate that proteotoxic stress triggers both non-canonical autophagy and TFEB-mediated canonical autophagy, with Atg8-family protein lipidation contributing to TFEB activation. Our results provide novel insights into how proteotoxic stress engages non-canonical MTORC1 inhibition and TFEB activation, thereby enhancing understanding of cellular adaptation to proteotoxic stress.: ALP, autophagy-lysosomal pathway; ATF4, activating transcription factor 4; Baf A1, bafilomycin A; CHX, cycloheximide; BTZ, bortezomib; CFZ, carfilzomib; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; DQ-BSA, dequenched-bovine serum albumin; EIF4EBP1/4EBP1, eukaryotic translation initiation factor 4E binding protein 1; ER, endoplasmic reticulum; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MG132, carbobenzoxy-Leu-Leu-leucinal; MTORC1, mechanistic target of rapamycin kinase complex 1; RPS6KB1/p70, ribosomal protein S6 kinase B1; RRAG, Ras related GTP binding; SQSTM1/p62, sequestosome 1; TFE3, transcription factor E3; TFEB, transcription factor EB; TSC2, TSC complex subunit 2; tfLC3, tandem fluorescent LC3; UPS, ubiquitin-proteasome system. ATG16L1ATG5Abbreviations1
