Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.

Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.

The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6.

The cellular activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but whether lysosomes contribute to this process remains unclear. Here, we show the vital role of the lysosomal membrane-tethered Ragulator complex in NLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophages and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed marked attenuation of NLRP3-associated inflammatory disease severity, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with both NLRP3 and histone deacetylase 6 (HDAC6). HDAC6 enhances the interaction between Lamtor1 and NLRP3, resulting in NLRP3 inflammasome activation. DL-all-rac-α-tocopherol, a synthetic form of vitamin E, inhibited the Lamtor1-HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. Further, DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. These results provide novel insights into the role of lysosomes in the activation of NLRP3 inflammasomes by the Ragulator complex.

A TGF-ß-responsive enhancer regulates SRC expression and epithelial-mesenchymal transition-associated cell migration.

The non-receptor tyrosine kinase SRC is overexpressed and/or hyperactivated in various human cancers, and facilitates cancer progression by promoting invasion and metastasis. However, the mechanisms underlying SRC upregulation are poorly understood. In this study, we demonstrate that transforming growth factor-ß (TGF-ß) induces SRC expression at the transcriptional level by activating an intragenic the SRC enhancer. In the human breast epithelial cell line MCF10A, TGF-ß1 stimulation upregulated one of the SRC promotors, the 1A promoter, resulting in increased SRC mRNA and protein levels. Chromatin immunoprecipitation (ChIP)-sequencing analysis revealed that the SMAD complex is recruited to three enhancer regions ∼15 kb upstream and downstream of the SRC promoter, and one of them is capable of activating the SRC promoter in response to TGF-ß. JUN, a member of the activator protein (AP)-1 family, localises to the enhancer and regulates TGF-ß-induced SRC expression. Furthermore, TGF-ß-induced SRC upregulation plays a crucial role in epithelial-mesenchymal transition (EMT)-associated cell migration by activating the SRC-focal adhesion kinase (FAK) circuit. Overall, these results suggest that TGF-ß-induced SRC upregulation promotes cancer cell invasion and metastasis in a subset of human malignancies.

Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids.

The mTORC1 kinase promotes growth in response to growth factors, energy levels, and amino acids, and its activity is often deregulated in disease. The Rag GTPases interact with mTORC1 and are proposed to activate it in response to amino acids by promoting mTORC1 translocation to a membrane-bound compartment that contains the mTORC1 activator, Rheb. We show that amino acids induce the movement of mTORC1 to lysosomal membranes, where the Rag proteins reside. A complex encoded by the MAPKSP1, ROBLD3, and c11orf59 genes, which we term Ragulator, interacts with the Rag GTPases, recruits them to lysosomes, and is essential for mTORC1 activation. Constitutive targeting of mTORC1 to the lysosomal surface is sufficient to render the mTORC1 pathway amino acid insensitive and independent of Rag and Ragulator, but not Rheb, function. Thus, Rag-Ragulator-mediated translocation of mTORC1 to lysosomal membranes is the key event in amino acid signaling to mTORC1.

The Ragulator complex serves as a substrate-specific mTORC1 scaffold in regulating the nuclear translocation of transcription factor EB.

The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by tenfold in the Ragulator-deficient clone compared with that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling.

SRC kinase activator CDCP1 promotes hepatocyte growth factor-induced cell migration/invasion of a subset of breast cancer cells.

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain-containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.

p18/Lamtor1-mTORC1 Signaling Controls Development of Mucin-producing Goblet Cells in the Intestine.

Mechanistic target of rapamycin complex 1 (mTORC1) plays a pivotal role in controlling cell growth and metabolism in response to nutrients and growth factors. The activity of mTORC1 is dually regulated by amino acids and growth factor signaling, and amino acid-dependent mTORC1 activity is regulated by mTORC1 interaction with the Ragulator-Rag GTPase complex, which is localized to the surface of lysosomes via a membrane-anchored protein, p18/Lamtor1. However, the physiological function of p18-Ragulator-dependent mTORC1 signaling remains elusive. The present study evaluated the function of p18-mediated mTORC1 signaling in the intestinal epithelia using p18 conditional knockout mice. In p18 knockout colonic crypts, mTORC1 was delocalized from lysosomes, and in vivo mTORC1 activity was markedly decreased. Histologically, p18 knockout crypts exhibited significantly increased proliferating cells and dramatically decreased mucin-producing goblet cells, while overall crypt architecture and enteroendocrine cell differentiation were unaffected. Furthermore, p18 knockout crypts normally expressed transcription factors implicated in crypt differentiation, such as Cdx2 and Klf4, indicating that p18 ablation did not affect the genetic program of cell differentiation. Analysis of colon crypt organoid cultures revealed that both p18 ablation and rapamycin treatment robustly suppressed development of mucin-producing goblet cells. Hence, p18-mediated mTORC1 signaling could promote the anabolic metabolism required for robust mucin production in goblet cells to protect the intestinal epithelia from various external stressors.Key words: mTORC1, p18/lamtor1, intestinal epithelium, goblet cells, mucin.

Rheb localized on the Golgi membrane activates lysosome-localized mTORC1 at the Golgi-lysosome contact site.

In response to amino acid supply, mTORC1, a master regulator of cell growth, is recruited to the lysosome and activated by the small GTPase Rheb. However, the intracellular localization of Rheb is controversial. In this study, we showed that a significant portion of Rheb is localized on the Golgi but not on the lysosome. GFP-Rheb could activate mTORC1, even when forced to exclusively localize to the Golgi. Likewise, artificial recruitment of mTORC1 to the Golgi allowed its activation. Accordingly, the Golgi was in contact with the lysosome at an newly discovered area of the cell that we term the Golgi-lysosome contact site (GLCS). The number of GLCSs increased in response to amino acid supply, whereas GLCS perturbation suppressed mTORC1 activation. These results suggest that inter-organelle communication between the Golgi and lysosome is important for mTORC1 regulation and the Golgi-localized Rheb may activate mTORC1 at GLCSs.

Ubiquitination of Src promotes its secretion via small extracellular vesicles.

Upregulation of the Src tyrosine kinase is implicated in the progression of cancer. The oncogenic potential of Src is suppressed via several negative regulation systems including degradation via the ubiquitin-proteasome pathway. Here, we show that ubiquitination of Src promotes its secretion via small extracellular vesicles (sEVs) to suppress its oncogenic potential. In MDCK cells expressing a modified Src that can be activated by hydroxytamoxifen, activated Src was transported to late endosomes/lysosomes and secreted via sEVs. The secretion of Src was suppressed by ablation of Cbl E3-ligase, suggesting the contribution of ubiquitination to this process. Activated Src was ubiquitinated at multiple sites, and Lys429 was identified as a critical site for sEV-mediated secretion. Mutation of Src at Lys429 (R429) caused resistance to ubiquitination and decreased its secretion via sEVs. The activated R429 mutant was also transported to late endosomes/lysosomes, whereas its incorporation into intraluminal vesicles was reduced. Activation of the R429 mutant induced a greater FAK activation than that of wild-type Src, thereby potentiating Src-induced invasive phenotypes, such as invadopodia formation and invasive activity. These findings demonstrate that ubiquitination of activated Src at Lys429 promotes its secretion via sEVs, suggesting a potential strategy to suppress the oncogenic function of upregulated Src.

Atg5-mediated autophagy controls apoptosis/anoikis via p53/Rb pathway in naked mole-rat fibroblasts.

The naked mole-rat (NMR, Heterocephalus glaber) is the longest-living known rodent species, with a maximum lifespan of over 30 years. NMRs exhibit negligible senescence, exceptional resistance to cancer, and high basal autophagy activity compared with mouse. The molecular mechanisms and physiological roles underlying the high basal autophagy activity in NMRs remain to be elucidated. We identified that the Atg12-Atg5 conjugate, a critical component of autophagosome formation, was highly expressed in NMR skin fibroblasts (NSFs) compared with that in mouse skin fibroblasts. Phenotypic analysis of Atg5 knockdown NSFs revealed that high basal autophagy activity in NSFs was associated with abundant expression of the Atg12-Atg5 conjugate. Atg5 knockdown in NSFs led to accumulation of dysfunctional mitochondria, and suppressed cell proliferation and cell adhesion ability, promoting apoptosis/anoikis accompanied by upregulation of the apoptosis-related genes, Bax and Noxa. Furthermore, inhibition of the p53/Rb pro-apoptotic pathway with SV40 large T antigen abolished Atg5 knockdown-induced increases in apoptosis/anoikis. Taken together, these findings suggest that high basal autophagy activity in NMR cells, mediated by Atg5, contributes to suppression of p53/Rb-induced apoptosis, which could benefit the longevity of NMR cells.

Lysosomal Protein Lamtor1 Controls Innate Immune Responses via Nuclear Translocation of Transcription Factor EB.

Amino acid metabolism plays important roles in innate immune cells, including macrophages. Recently, we reported that a lysosomal adaptor protein, Lamtor1, which serves as the scaffold for amino acid-activated mechanistic target of rapamycin complex 1 (mTORC1), is critical for the polarization of M2 macrophages. However, little is known about how Lamtor1 affects the inflammatory responses that are triggered by the stimuli for TLRs. In this article, we show that Lamtor1 controls innate immune responses by regulating the phosphorylation and nuclear translocation of transcription factor EB (TFEB), which has been known as the master regulator for lysosome and autophagosome biogenesis. Furthermore, we show that nuclear translocation of TFEB occurs in alveolar macrophages of myeloid-specific Lamtor1 conditional knockout mice and that these mice are hypersensitive to intratracheal administration of LPS and bleomycin. Our observation clarified that the amino acid-sensing pathway consisting of Lamtor1, mTORC1, and TFEB is involved in the regulation of innate immune responses.

Src mediates TGF-ß-induced intraocular pressure elevation in glaucoma.

Glaucoma, a progressive and irreversible optic neuropathy, is one of the leading causes of vision impairment worldwide. Elevation of intraocular pressure (IOP) due to transforming growth factor-ß (TGF-ß)-induced dysfunction of the trabecular meshwork is a risk factor for glaucoma, but the underlying molecular mechanisms remain elusive. Here, we show that Src kinase is involved in TGF-ß-induced IOP elevation. We observed that dasatinib, a potent Src inhibitor, suppressed TGF-ß2-induced IOP in rat eyes. Mechanistic analyses in human trabecular meshwork cells showed that TGF-ß2 activated Src signaling and concomitantly increased cytoskeletal remodeling, cell adhesion, and extracellular matrix (ECM) accumulation. Src was activated via TGF-ß2-induced upregulation of the Src scaffolding protein CasL, which mediates the assembly of focal adhesions, cytoskeletal remodeling, and ECM deposition. Activation of Src suppressed the expression of tissue plasminogen activator, thereby attenuating ECM degradation. Furthermore, the Src inhibitor ameliorated TGF-ß2-induced changes in the contractile and adhesive characteristics of trabecular meshwork cells, and ECM deposition. These findings underscore the crucial role of Src activity in TGF-ß-induced IOP elevation and identify Src signaling as a potential therapeutic target in glaucoma.

Lamtor1 Is Critically Required for CD4+ T Cell Proliferation and Regulatory T Cell Suppressive Function.

Mechanistic target of rapamycin complex (mTORC)1 integrates intracellular sufficiency of nutrients and regulates various cellular functions. Previous studies using mice with conditional knockout of mTORC1 component proteins (i.e., mTOR, Raptor, and Rheb) gave conflicting results on the roles of mTORC1 in CD4+ T cells. Lamtor1 is the protein that is required for amino acid sensing and activation of mTORC1; however, the roles of Lamtor1 in T cells have not been investigated. In this article, we show that Lamtor1-deficient CD4+ T cells exhibited marked reductions in proliferation, IL-2 production, mTORC1 activity, and expression of purine- and lipid-synthesis genes. Polarization of Th17 cells, but not Th1 and Th2 cells, diminished following the loss of Lamtor1. Accordingly, CD4-Cre-driven Lamtor1-knockout mice exhibited reduced numbers of CD4+ and CD8+ T cells at rest, and they were completely resistant to experimental autoimmune encephalomyelitis. In contrast, genetic ablation of Lamtor1 in Foxp3+ T cells resulted in severe autoimmunity and premature death. Lamtor1-deficient regulatory T cells survived ex vivo as long as wild-type regulatory T cells; however, they exhibited a marked loss of suppressive function and expression of signature molecules, such as CTLA-4. These results indicate that Lamtor1 plays essential roles in CD4+ T cells. Our data suggest that Lamtor1 should be considered a novel therapeutic target in immune systems.

Role of Ragulator in the Regulation of Mechanistic Target of Rapamycin Signaling in Podocytes and Glomerular Function.

Aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1) in glomerular podocytes leads to glomerular insufficiency and may contribute to the development of glomerular diseases, including diabetic nephropathy. Thus, an approach for preventing mTORC1 activation may allow circumvention of the onset and progression of mTORC1-dependent podocyte injury and glomerular diseases. mTORC1 activation requires inputs from both growth factors and nutrients that inactivate the tuberous sclerosis complex (TSC), a key suppressor of mTORC1, on the lysosome. Previous studies in mice revealed that the growth factor-phosphatidylinositol 3-kinase pathway and mTORC1 are essential for maintaining normal podocyte function, suggesting that direct inhibition of the phosphatidylinositol 3-kinase pathway or mTORC1 may not be an ideal approach to sustaining physiologic podocyte functions under certain disease conditions. Here, we report the role of the Ragulator complex, which recruits mTORC1 to lysosomes in response to nutrient availability in podocytes. Notably, podocytes lacking Ragulator maintain basal mTORC1 activity. Unlike podocyte-specific mTORC1-knockout mice, mice lacking functional Ragulator in podocytes did not show abnormalities in podocyte or glomerular function. However, aberrant mTORC1 activation induced by active Rheb in podocyte-specific TSC1-knockout (podo-TSC1 KO) mice did require Ragulator. Moreover, ablation of Ragulator in the podocytes of podo-TSC1 KO mice or streptozotocin-induced diabetic mice significantly blocked the development of pathologic renal phenotypes. These observations suggest that the blockade of mTORC1 recruitment to lysosomes may be a useful clinical approach to attenuate aberrant mTORC1 activation under certain disease conditions.

The lipid raft-anchored adaptor protein Cbp controls the oncogenic potential of c-Src.

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.

The lysosomal signaling anchor p18/LAMTOR1 controls epidermal development by regulating lysosome-mediated catabolic processes.

The lysosomal adaptor protein p18 is an essential anchor of a scaffolding complex for the mTORC1 and MAPK pathways, which play crucial roles in controlling cell growth and energy homeostasis. To elucidate the in vivo function of the p18-mediated pathway, we conditionally ablated p18 in the mouse epidermis. Mutant mice were born with severe defects in formation of the stratum corneum and died within 12 h after birth due to dehydration caused by loss of skin barrier function. Mutant epidermal cells can grow and differentiate into granular cells, but exhibit functional defects in corneocyte maturation. Electron microscopy identified abnormal immature cells, overlying the mutant granular cells, which accumulated autophagosomes, glycogen granules and dead nuclei. Cell culture analysis showed that loss of p18 attenuated lysosome function, resulting in accumulation of immature lysosomes and autophagosomes. Analyses of lysosome behavior revealed that p18 is required for functional interaction between lysosomes and target organelles including autophagosomes. These findings suggest that p18-mediated pathways control lysosome-mediated catabolic processes, which are crucial for the development of mouse epidermis.

Csk restrains BCR-mediated ROS production and contributes to germinal center selection and affinity maturation.

Compared with naïve B cells, the B cell receptor (BCR) signal in germinal center (GC) B cells is attenuated; however, the significance of this signaling attenuation has not been well defined. Here, to investigate the role of attenuation of BCR signaling, we employed a Csk mutant mouse model in which Csk deficiency in GC B cells resulted in augmentation of net BCR signaling with no apparent effect on antigen presentation. We found that Csk is required for GC maintenance and efficient antibody affinity maturation. Mechanistically, ROS-induced apoptosis was exacerbated concomitantly with mitochondrial dysfunction in Csk-deficient GC B cells. Hence, our data suggest that attenuation of the BCR signal restrains hyper-ROS production, thereby protecting GC B cells from apoptosis and contributing to efficient affinity maturation.

The guanine nucleotide exchange factor Arhgef5 plays crucial roles in Src-induced podosome formation.

Podosomes and invadopodia are actin-rich membrane protrusions that play a crucial role in cell adhesion and migration, and extracellular matrix remodeling in normal and cancer cells. The formation of podosomes and invadopodia is promoted by upregulation of some oncogenic molecules and is closely related to the invasive potential of cancer cells. However, the molecular mechanisms underlying the podosome and invadopodium formation still remain unclear. Here, we show that a guanine nucleotide exchange factor (GEF) for Rho family GTPases (Arhgef5) is crucial for Src-induced podosome formation. Using an inducible system for Src activation, we found that Src-induced podosome formation depends upon the Src SH3 domain, and identified Arhgef5 as a Src SH3-binding protein. RNA interference (RNAi)-mediated depletion of Arhgef5 caused robust inhibition of Src-dependent podosome formation. Overexpression of Arhgef5 promoted actin stress fiber remodeling through activating RhoA, and the activation of RhoA or Cdc42 was required for Src-induced podosome formation. Arhgef5 was tyrosine-phosphorylated by Src and bound to Src to positively regulate its activity. Furthermore, the pleckstrin homology (PH) domain of Arhgef5 was required for podosome formation, and Arhgef5 formed a ternary complex with Src and phosphoinositide 3-kinase when Src and/or Arhgef5 were upregulated. These findings provide novel insights into the molecular mechanisms of podosome and invadopodium formation induced by Src upregulation.

Redox regulates mammalian target of rapamycin complex 1 (mTORC1) activity by modulating the TSC1/TSC2-Rheb GTPase pathway.

Mammalian target of rapamycin (mTOR) is a kinase that plays a key role in a wide array of cellular processes and exists in two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although mTORC2 is primarily activated by growth factors, mTORC1 is regulated by numerous extracellular and intracellular signals such as nutrients, growth factors, and cellular redox. Previous study has shown that cysteine oxidants sufficiently activate mTORC1 activity under amino acid-depleted conditions and that a reducing agent effectively suppresses amino acid-induced mTORC1 activity, thereby raising the possibility that redox-sensitive mechanisms underlie amino acid-dependent mTORC1 regulation. However, the molecular mechanism by which redox regulates mTORC1 activity is not well understood. In this study, we show that the redox-sensitive regulation of mTORC1 occurs via Rheb but not the Rag small GTPase. Enhancing cellular redox potential with cysteine oxidants significantly increases Rheb GTP levels. Importantly, modulation of the cellular redox potential with a cysteine oxidant or reducing agent failed to alter mTORC1 activity in TSC1(-/-) or TSC2(-/-) mouse embryonic fibroblast cells. Furthermore, a cysteine oxidant has little effect on mTOR localization but sufficiently activates mTORC1 activity in both p18(-/-) and control mouse embryonic fibroblast cells, suggesting that the redox-sensitive regulation of mTORC1 occurs independent of the Ragulator·Rag complex. Taken together, our results suggest that the TSC complex plays an important role in redox-sensitive mTORC1 regulation and argues for the activation of mTORC1 in places other than the lysosome upon inhibition of the TSC complex.

The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes.

The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.