Organelles in eukaryotic cells are compartmentalized to carry out different functions, and require specific mechanisms governing their coordination. Two of these organelles, the lysosome and the endoplasmic reticulum (ER), play crucial roles in regulating cellular homeostasis and organismal health. The lysosome contains various enzymes devoted to the hydrolysis of specific substrates, and dysfunctions of these hydrolases and their related metabolic processes are implicated in many diseases 1 . On the other hand, ER is essential for protein synthesis and utilizes quality control mechanisms to maintain proteostasis. The metabolic status of the lysosome is now known to actively influence nuclear transcription and mitochondrial signaling 2-4 . However, whether and how mechanistically lysosomal metabolic activities regulate ER proteostasis remain unclear. Here, we reported that RSH-1, the Caenorhabditis elegans RelA/SpoT Homolog (RSH) protein, carries a lysosomal NADPH phosphatase activity and acts through the mammalian/mechanistic target of rapamycin (mTOR) signaling to regulate ER proteostasis. We discovered that RSH-1 is localized to the lysosome. Its mutation reduces NADPH hydrolysis by the lysosome, leading to a protection against ER stress-induced lethality. We further revealed that this ER stress tolerance requires lysosomal vacuolar-type H + -ATPase (v-ATPase), and Rag GTPases and ribosomal protein S6 kinase (S6K) in the mTOR signaling pathway. Through transcriptome analysis, we discovered that the S6K activation by the RSH-1 mutation increases the basal expression of X-box binding protein 1 (XBP-1) and peptidyl-prolyl cis-trans isomerases, which is necessary for improving ER proteostasis. Moreover, we demonstrated the lysosomal localization of RSH-1 mammalian homolog and its conserved role in regulating mTOR signaling. Together, our findings reveal the key role of a specific lysosomal nucleotide hydrolase in regulating organellar coordination and also signaling mechanisms underlying this active regulation, and suggest that diseases resulted from deficiency in lysosomal hydrolases may be attributed to the distortion of signal transduction and organelle homeostasis.RSHs are a superfamily of proteins that are well conserved across evolution in bacteria, plants, and metazoa 5 , and regulate growth and stress responses. The RSH family of proteins in bacteria and plants carries both synthetase and hydrolase domains (Fig. 1a) that are known to synthesize and hydrolyze (p)ppGpp, a GDP/GTP-derived nucleotide messenger, respectively 6-9 . On the other hand, the metazoan RSH carries only a hydrolase domain (Fig. 1a), and can dephosphorylate ppGpp and NADPH in vitro 8,10 . Drosophila RSH homolog regulates starvation response 8 , while the human homolog facilitates ferroptosis 10 . In plants, RSH catalyzes (p)ppGpp metabolism within a well-defined localization in the chloroplast, an eukaryotic organelle with a bacterial origin 7,9,11 . We thus hypothesized that the metazoan RSH might also have a specialized subcellular co...
