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Amino acid availability controls mTORC1 activity via a heterodimeric Rag GTPase complex that functions as a scaffold at the lysosomal surface, bringing together mTORC1 with its activators and effectors. Mammalian cells express four Rag proteins (RagA–D) that form dimers composed of RagA/B bound to RagC/D. Traditionally, the Rag paralogue pairs (RagA/B and RagC/D) are referred to as functionally redundant, with the four dimer combinations used interchangeably in most studies. Here, by using genetically modified cell lines that express single Rag heterodimers, we uncover a Rag dimer code that determines how amino acids regulate mTORC1. First, RagC/D differentially define the substrate specificity downstream of mTORC1, with RagD promoting phosphorylation of its lysosomal substrates TFEB/TFE3, while both Rags are involved in the phosphorylation of non-lysosomal substrates such as S6K. Mechanistically, RagD recruits mTORC1 more potently to lysosomes through increased affinity to the anchoring LAMTOR complex. Furthermore, RagA/B specify the signalling response to amino acid removal, with RagB-expressing cells maintaining lysosomal and active mTORC1 even upon starvation. Overall, our findings reveal key qualitative differences between Rag paralogues in the regulation of mTORC1, and underscore Rag gene duplication and diversification as a potentially impactful event in mammalian evolution.

Changes in splicing fidelity are associated with loss of homeostasis and ageing1–3, yet only a handful of splicing factors have been shown to be causally required to promote longevity 1–3, and the underlying mechanisms and downstream targets in these paradigms remain elusive. Surprisingly, we found a hypomorphic mutation within RNP-6/PUF60, a spliceosome component promoting weak 3’ splice site recognition, which causes aberrant splicing, elevated stress responses, and enhances longevity in Caenorhabditis elegans. Through genetic suppressor screens, we identify a gain-of-function mutation within rbm-39, an RNP-6 interacting splicing factor, which increases nuclear speckle formation, alleviates splicing defects and curtails longevity caused by rnp-6 mutation. By leveraging the splicing changes induced by RNP-6/RBM-39 activities, we uncover a single intron retention event in egl-8/phospholipase C B4 as a key splicing target prolonging life. Genetic and biochemical evidence show that neuronal RNP-6/EGL-8 downregulate mTORC1 signaling to control organismal life span. In mammalian cells, PUF60 downregulation also potently and specifically inhibits mTORC1 signaling. Altogether, our results reveal that splicing fidelity modulates mTOR signaling and suggest a potential therapeutic strategy to delay ageing.

Cell growth is regulated primarily by the mammalian/mechanistic Target of Rapamycin Complex 1 (mTORC1) that functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways 1. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid (FA) biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here, we show that mTORC1 senses the capacity of a cell to synthesize FAs by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is very direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When ACC1 (acetyl-CoA carboxylase 1) is hyperactive or FASN (fatty acid synthase) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with ACC1 and FASN. Our findings represent a conserved, unique, homeostatic mechanism whereby impaired FA biogenesis leads to reduced mTORC1 activity to coordinatively link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the first-described example of a physiological metabolite that directly inhibits the activity of a signalling kinase by competing with ATP for binding.Competing Interest StatementThe authors have declared no competing interest.

Rapamycin is a macrolide antibiotic that functions as an immunosuppressive and anti-cancer agent, and displays robust anti-ageing effects in multiple organisms including humans. Importantly, rapamycin analogs (rapalogs) are of clinical importance against certain cancer types and neurodevelopmental diseases. Although rapamycin is widely perceived as an allosteric inhibitor of mTOR (mechanistic target of rapamycin), the master regulator of cellular and organismal physiology, its specificity has not been thoroughly evaluated so far. In fact, previous studies in cells and in mice suggested that rapamycin may be also acting independently from mTOR to influence various cellular functions. Here, we generated a gene-edited cell line, that expresses a rapamycin-resistant mTOR mutant (mTORRR), and assessed the effects of rapamycin treatment on the transcriptome and proteome of control or mTORRR-expressing cells. Our data reveal a striking specificity of rapamycin towards mTOR, demonstrated by virtually no changes in mRNA or protein levels in rapamycin-treated mTORRRcells, even following prolonged drug treatment. Overall, this study provides the first comprehensive and conclusive assessment of rapamycin’s specificity, with important potential implications for ageing research and human therapeutics.

The mechanistic Target of Rapamycin (mTOR) is a growth-related kinase that, in the context of the mTOR complex 1 (mTORC1), touches upon most fundamental cellular processes. Consequently, its activity is a critical determinant for cellular and organismal physiology, while its dysregulation is commonly linked to human aging and age-related disease. Presumably the most important stimulus that regulates mTORC1 activity is nutrient sufficiency, whereby amino acids play a predominant role. In fact, mTORC1 functions as a molecular sensor for amino acids, linking the cellular demand to the nutritional supply. Notably, dietary restriction (DR), a nutritional regimen that has been shown to extend lifespan and improve healthspan in a broad spectrum of organisms, works via limiting nutrient uptake and changes in mTORC1 activity. Furthermore, pharmacological inhibition of mTORC1, using rapamycin or its analogs (rapalogs), can mimic the pro-longevity effects of DR. Conversely, nutritional amino acid overload has been tightly linked to aging and diseases, such as cancer, type 2 diabetes and obesity. Similar effects can also be recapitulated by mutations in upstream mTORC1 regulators, thus establishing a tight connection between mTORC1 signaling and aging. Although the role of growth factor signaling upstream of mTORC1 in aging has been investigated extensively, the involvement of signaling components participating in the nutrient sensing branch is less well understood. In this review, we provide a comprehensive overview of the molecular and cellular mechanisms that signal nutrient availability to mTORC1, and summarize the role that nutrients, nutrient sensors, and other components of the nutrient sensing machinery play in cellular and organismal aging.

Molecular cell 81(13), 2705 - 2721.e8 (2021). doi:10.1016/j.molcel.2021.04.019, The TSC complex is a critical negative regulator of the small GTPase Rheb and mTORC1 in cellular stress signaling. The TSC2 subunit contains a catalytic GTPase activating protein domain and interacts with multiple regulators, while the precise function of TSC1 is unknown. Here we provide a structural characterization of TSC1 and define three domains: a C-terminal coiled-coil that interacts with TSC2, a central helical domain that mediates TSC1 oligomerization, and an N-terminal HEAT repeat domain that interacts with membrane phosphatidylinositol phosphates (PIPs). TSC1 architecture, oligomerization, and membrane binding are conserved in fungi and humans. We show that lysosomal recruitment of the TSC complex and subsequent inactivation of mTORC1 upon starvation depend on the marker lipid PI3,5P2, demonstrating a role for lysosomal PIPs in regulating TSC complex and mTORC1 activity via TSC1. Our study thus identifies a vital role of TSC1 in TSC complex function and mTORC1 signaling., Published by Elsevier, New York, NY

Summary Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling., Graphical Abstract, Highlights • G3BPs act non-redundantly in the TSC-mTORC1 signaling axis • G3BPs reside at the lysosomal surface and inhibit mTORC1 • The TSC complex requires G3BPs as its lysosomal tether • G3BP1 deficiency phenocopies TSC complex loss in cancer cells and neurons, Distinct from their contributions to stress granules, G3BPs regulate mTORC1 activity through spatial control of the TSC complex.

SummaryG3BP1 (Ras GTPase-activating protein-binding protein 1) is widely recognized as a core component of stress granules (SG), non-membranous RNA-protein-assemblies required for cellular survival under stress. We report that in the absence of SG, G3BP1 acts as lysosomal anchor of the Tuberous Sclerosis Complex (TSC) protein complex. By tethering the TSC complex to lysosomes, G3BP1 suppresses signaling through the metabolic master regulator mTORC1 (mechanistic target of rapamycin complex 1). Like the known TSC complex subunits, G3BP1 suppresses phenotypes related to mTORC1 hyperactivity in the context of tumors and neuronal dysfunction. Thus, G3BP1 is not only a core component of SG but also a key element of lysosomal TSC-mTORC1 signaling.HighlightsThebona fidestress granule component G3BP1is a key element of the TSC-mTORC1 signaling axis.tethers the TSC complex to lysosomes.prevents mTORC1 hyperactivation by metabolic stimuli.suppresses mTORC1-driven cancer cell motility and epileptiform activity.Graphical Abstract

SummaryTOR complex 1 (TORC1) is a potent anabolic regulator of cellular growth and metabolism. When cells have sufficient amino acids, TORC1 is active due to its lysosomal localization mediated via the Rag GTPases. Upon amino acid removal, the Rag GTPases release TORC1, causing it to become cytoplasmic and inactive. We show here that, upon amino acid removal, the Rag GTPases also recruit TSC2 to the lysosome, where it can act on Rheb. Only when both the Rag GTPases and Rheb are inactive is TORC1 fully released from the lysosome. Upon amino acid withdrawal, cells lacking TSC2 fail to completely release TORC1 from the lysosome, fail to completely inactivate TORC1, and fail to adjust physiologically to amino acid starvation. These data suggest that regulation of TSC2 subcellular localization may be a general mechanism to control its activity and place TSC2 in the amino-acid-sensing pathway to TORC1.

Amino acids regulate TOR complex 1 (TORC1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC1 recruitment to lysosomes where TORC1 is activated by binding Rheb. How the absence of amino acids inactivates TORC1 is less well understood. Amino acid starvation recruits the TSC1/TSC2 complex to the vicinity of TORC1 to inhibit Rheb; however, the upstream mechanisms regulating TSC2 are not known. We identify here the eIF4A-containing eIF4F translation initiation complex as an upstream regulator of TSC2 in response to amino acid withdrawal in Drosophila. We find that TORC1 and translation preinitiation complexes bind each other. Cells lacking eIF4F components retain elevated TORC1 activity upon amino acid removal. This effect is specific for eIF4F and not a general consequence of blocked translation. This study identifies specific components of the translation machinery as important mediators of TORC1 inactivation upon amino acid removal.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a dominant oncoprotein that has been implicated in the development of most EBV-associated malignancies (reviewed in reference 28). LMP1 is an integral membrane protein that consists of a short amino-terminal cytoplasmic region, six transmembrane domains, and a 200-amino-acid cytoplasmic carboxyl-terminal tail (CCT). It signals continuously by virtue of its intrinsic ability to oligomerize and interact constitutively with intracellular signaling molecules (reviewed in reference 19). It mimics activated members of the tumor necrosis factor receptor family to transmit growth and antiapoptotic signals. These signals are conveyed to the nucleus through the activation of the canonical and noncanonical NF-κB activation pathways as well as mitogen-activated protein kinase pathways. The activation of NF-κB by LMP1 is of particular importance since it is essential for B-lymphocyte transformation by EBV (6, 7, 10). Therefore, the mechanism of NF-κB activation by LMP1 and its effects on specific cellular gene expression have been the subject of intense investigation. Two regions in the CCT of LMP1 mediate the activation of NF-κB (reviewed in reference 33). The membrane-proximal region CTAR1 mediates primarily the activation of the noncanonical NF-κB activation pathway and coincides with the principal transformation effector site (TES1). A second region (CTAR2), which is located near the carboxyl terminus of the CCT, mediates a powerful induction of the canonical NF-κB activation pathway and coincides with a second transformation effector site (TES2). The role of LMP1-induced NF-κB activation in cellular gene induction has been demonstrated in many cases. However, until recently, NF-κB has not been implicated in the transactivation of EBV genes. The expression of LMP1 is suppressed in EBV-infected B lymphocytes of healthy carriers and EBV-positive Burkitt's lymphomas. Nevertheless, LMP1 expression is permissive in most EBV-associated malignancies, and for this reason, the mechanism of LMP1 transcription regulation has been the subject of intense investigation. Two promoters can drive the transcription of the LMP1 gene (reviewed in reference 19). A proximal promoter (ED-L1) is active in latency III, whereas a distal promoter (LT-R1) located in the terminal repeats mediates the expression of LMP1 in latency II. A number of viral and cellular transcription regulators have been implicated in the regulation of the ED-L1 LMP1 promoter (which will be referred to as the LMP1 promoter from this point on) using primarily EBV-negative B-cell lymphoma and epithelial cell lines as reporter systems. These experiments have shown a prominent role of the latent EBV antigens EBNA2 and EBNALP in the transactivation of the LMP1 gene, which depends on the RBPJκ, PU.1, POU-box, and AP2 binding sites of the LMP1 promoter (11, 14, 15, 26, 27, 31, 36, 39). The activity of the LMP1 promoter can be regulated also by the latent EBV antigens EBNA3A and EBNA3C (29). In addition, activating transcription factor/cyclic AMP response element (ATF/CRE), interferon regulatory factor (IRF), and STAT elements have been implicated in the transactivation of the LMP1 promoter (13, 24, 25, 32). In the present report, bioinformatic tools, macromolecular interaction analyses, and transcription reporter assays identified a direct mechanism of regulation of LMP1 transcription by NF-κB and established the role of several other cis-regulatory elements of the LMP1 promoter in type III latent infection of B lymphocytes by EBV.

mTORC1 promotes cell growth and is therefore inactivated upon unfavourable growth conditions. Signalling pathways downstream of most cellular stresses converge on TSC1/2, which serves as an integration point that inhibits mTORC1. The TSC1/2 complex was shown to translocate to lysosomes to inactivate mTORC1 in response to two stresses: amino-acid starvation and growth factor removal. Whether other stresses also regulate TSC2 localization is not known. How TSC2 localization responds to combinations of stresses and other stimuli is also unknown. We show that both amino acids and growth factors are required simultaneously to maintain TSC2 cytoplasmic; when one of the two is missing, TSC2 relocalizes to lysosomes. Furthermore, multiple different stresses that inhibit mTORC1 also drive TSC2 lysosomal accumulation. Our findings indicate that lysosomal recruitment of TSC2 is a universal response to stimuli that inactivate mTORC1, and that the presence of any single stress is sufficient to cause TSC2 lysosomal localization., In response to amino acid and growth factor removal the TSC1/2 complex translocates to the lysosome to inactivate mTOR and inhibit cell growth. Here, the authors have shown that other cellular stresses also trigger this translocation to the lysosome suggesting that this is a universal mechanism in the stress response.

mTOR complex 1 (mTORC1) regulates cell growth and metabolism. mTORC1 activity is regulated via integration of positive growth-promoting stimuli and negative stress stimuli. One stress cells confront in physiological and pathophysiological contexts is hyperosmotic stress. The mechanism by which hyperosmotic stress regulates mTORC1 activity is not well understood. We show here that mild hyperosmotic stress induces a rapid and reversible inactivation of mTORC1 via a mechanism involving multiple upstream signaling pathways. We find that hyperosmotic stress causes dynamic changes in TSC2 phosphorylation by upstream kinases, such as Akt, thereby recruiting TSC2 from the cytoplasm to lysosomes where it acts on Rheb, the direct activator of mTORC1. This work puts together a signaling pathway whereby hyperosmotic stress inactivates mTORC1.

Hepatocyte nuclear factor-4 (HNF-4α), a member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer. Dimerization controls important aspects of receptor function, such as DNA binding, protein stability, ligand binding and interaction with coactivators. Crystallographic data of the HNF-4α ligand-binding domain (LBD) demonstrated that the homodimer interface is composed of residues in helices 7, 9 and 10 with intermolecular salt bridges, hydrogen bonds and hydrophobic interactions contributing to the stability of the interface. To investigate the importance of the proposed ionic interactions for HNF-4α dimerization, interactions critical for formation of the LBD homodimer interface were disrupted by introducing point mutations in residues D261N (H7), E269Q (H7), Q307L (H9), D312N (H9) and Q336L (H10). Mutants were analysed for transactivation, coactivator interaction, DNA binding and dimerization. EMSA analysis showed that the mutants are able to bind DNA as dimers and coimmunoprecipitation assays confirmed dimerization in solution. Furthermore, the mutations do not compromise HNF-4α activity and are responsive to PPAR-gamma coactivator-1 (PGC-1). Finally, residue R324, located in the H9/H10 loop, which was suspected to be involved in dimer stabilization via an ionic interaction with residue E276, was studied. In contrast to the conservative substitution R324H the mutation R324L abolishes HNF-4α transcriptional activity and coactivator recruitment, revealing that the nature of substitution may play an important role in HNF-4α function.

Summary The molecular mechanisms regulating animal tissue size during development are unclear. This question has been extensively studied in the Drosophila wing disc. Although cell growth is regulated by the kinase TORC1, no readout exists to visualize TORC1 activity in situ in Drosophila . Both the cell cycle and the morphogen Dpp are linked to tissue growth, but whether they regulate TORC1 activity is not known. We develop here an anti-phospho-dRpS6 antibody that detects TORC1 activity in situ . We find, unexpectedly, that TORC1 activity in the wing disc is patchy. This is caused by elevated TORC1 activity at the cell cycle G 1 /S transition due to CycD/Cdk4 phosphorylating TSC1/2. We find that TORC1 is also activated independently of CycD/Cdk4 when cells with different levels of Dpp signaling or Brinker protein are juxtaposed. We thereby characterize the spatial distribution of TORC1 activity in a developing organ.

The transcription factor NF-kappaB is a critical regulator of immune responses. To determine how NF-kappaB builds transcriptional control networks, we need to obtain a topographic map of the factor bound to the genome and correlate it with global gene expression. We used a ChIP cloning technique and identified novel NF-kappaB target genes in response to virus infection. We discovered that most of the NF-kappaB-bound genomic sites deviate from the consensus and are located away from conventional promoter regions. Remarkably, we identified a novel abundant NF-kappaB-binding site residing in specialized Alu-repetitive elements having the potential for long range transcription regulation, thus suggesting that in addition to its known role, NF-kappaB has a primate-specific function and a role in human evolution. By combining these data with global gene expression profiling of virus-infected cells, we found that most of the sites bound by NF-kappaB in the human genome do not correlate with changes in gene expression of the nearby genes and they do not appear to function in the context of synthetic promoters. These results demonstrate that repetitive elements interspersed in the human genome function as common target sites for transcription factors and may play an important role in expanding the repertoire of binding sites to engage new genes into regulatory networks.

Hepatocyte nuclear factor-4 (HNF-4alpha), a member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer. Dimerization controls important aspects of receptor function, such as DNA binding, protein stability, ligand binding and interaction with coactivators. Crystallographic data of the HNF-4alpha ligand-binding domain (LBD) demonstrated that the homodimer interface is composed of residues in helices 7, 9 and 10 with intermolecular salt bridges, hydrogen bonds and hydrophobic interactions contributing to the stability of the interface. To investigate the importance of the proposed ionic interactions for HNF-4alpha dimerization, interactions critical for formation of the LBD homodimer interface were disrupted by introducing point mutations in residues D261N (H7), E269Q (H7), Q307L (H9), D312N (H9) and Q336L (H10). Mutants were analysed for transactivation, coactivator interaction, DNA binding and dimerization. EMSA analysis showed that the mutants are able to bind DNA as dimers and coimmunoprecipitation assays confirmed dimerization in solution. Furthermore, the mutations do not compromise HNF-4alpha activity and are responsive to PPAR-gamma coactivator-1 (PGC-1). Finally, residue R324, located in the H9/H10 loop, which was suspected to be involved in dimer stabilization via an ionic interaction with residue E276, was studied. In contrast to the conservative substitution R324H the mutation R324L abolishes HNF-4alpha transcriptional activity and coactivator recruitment, revealing that the nature of substitution may play an important role in HNF-4alpha function.

Hepatocyte nuclear factor-4 (HNF-4) is a transcription factor of the nuclear hormone receptor superfamily that is constitutively active without the addition of exogenous ligand. Crystallographic analysis of the HNF-4alpha and HNF-4gamma ligand binding domains (LBDs) demonstrated the presence of endogenous ligands that may act as structural cofactors for HNF-4. It was also proposed by crystallographic studies that a combination of ligand and coactivator might be required to lock the receptor in its active state. We previously showed that mutations in amino acid residues Ser-181 and Met-182 in H3, Leu-219 and Leu-220 and Arg-226 in H5, Ileu-338 in H10, and Ileu-346 in H11, which line the LBD pocket in HNF-4alpha and come in contact with the ligand, impair its transactivation potential. In the present study, physical and functional interaction assays were utilized with two different coactivators, PGC-1 and SRC-3, to address the role of coactivators in HNF-4 function. We show that the integrity of the hinge (D) domain of HNF-4alpha and the activation function (AF)-2 activation domain region are critical for coactivation. Surprisingly, a different mode of coactivation is observed among the LBD point mutants that lack transcriptional activity. In particular, coactivation is maintained in mutants Ser-181, Arg-226, and Ile-346 but is abolished in mutants Met-182, Leu-219, and Ile-338. Physical interactions confirm this pattern of activation, implying that distinct amino acid residues may be involved in coactivator and ligand interactions, although some residues may be critical for both functions. Our results provide evidence and expand predictions based on the crystallographic data as to the role of coactivators in HNF-4alpha constitutive transcriptional activity.

Changes in splicing fidelity are associated with loss of homeostasis and aging, yet only a handful of splicing factors have been shown to be causally required to promote longevity, and the underlying mechanisms and downstream targets in these paradigms remain elusive. Surprisingly, we found a hypomorphic mutation within ribonucleoprotein RNP-6/poly(U)-binding factor 60 kDa (PUF60), a spliceosome component promoting weak 3′-splice site recognition, which causes aberrant splicing, elevates stress responses and enhances longevity in Caenorhabditis elegans. Through genetic suppressor screens, we identify a gain-of-function mutation within rbm-39, an RNP-6-interacting splicing factor, which increases nuclear speckle formation, alleviates splicing defects and curtails longevity caused by rnp-6 mutation. By leveraging the splicing changes induced by RNP-6/RBM-39 activities, we uncover intron retention in egl-8/phospholipase C β4 (PLCB4) as a key splicing target prolonging life. Genetic and biochemical evidence show that neuronal RNP-6/EGL-8 downregulates mammalian target of rapamycin complex 1 (mTORC1) signaling to control organismal lifespan. In mammalian cells, PUF60 downregulation also potently and specifically inhibits mTORC1 signaling. Altogether, our results reveal that splicing fidelity modulates lifespan through mTOR signaling.

Cellular adaptation to stress is a crucial homeostatic process for survival, metabolism, physiology, and disease. Cells respond to stress stimuli (e.g., nutrient starvation, growth factor deprivation, hypoxia, low energy, etc.) by changing the activity of signaling pathways, and interact with their environment by qualitatively and quantitatively modifying their intracellular, surface, and extracellular proteomes. How this delicate communication takes place is a hot topic in cell biological research, and has important implications for human disease.

Summary Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress., Graphical abstract, Highlights • mTORC1 phosphorylates GRASP55 directly at the Golgi in non-stressed cells • mTORC1 inactivation by stress leads to GRASP55 dephosphorylation and relocalization • GRASP55 relocalization to autophagosomes and MVBs drives UPS of selected cargo • mTORC1-GRASP55 link cellular stress to changes in the extracellular proteome via UPS, Cells adapt to stress stimuli by qualitatively and quantitatively reshaping their extracellular proteome. Nüchel et al. highlight the biological role of unconventional protein secretion (UPS) and reveal TSC-mTORC1-GRASP55 as a key signaling pathway in this process, thus linking physiological stress signaling and nutrient sensing to the cellular stress response.