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1.
Cell ; 179(6): 1319-1329.e8, 2019 11 27.
Article in English | MEDLINE | ID: mdl-31704029

ABSTRACT

mTORC1 controls anabolic and catabolic processes in response to nutrients through the Rag GTPase heterodimer, which is regulated by multiple upstream protein complexes. One such regulator, FLCN-FNIP2, is a GTPase activating protein (GAP) for RagC/D, but despite its important role, how it activates the Rag GTPase heterodimer remains unknown. We used cryo-EM to determine the structure of FLCN-FNIP2 in a complex with the Rag GTPases and Ragulator. FLCN-FNIP2 adopts an extended conformation with two pairs of heterodimerized domains. The Longin domains heterodimerize and contact both nucleotide binding domains of the Rag heterodimer, while the DENN domains interact at the distal end of the structure. Biochemical analyses reveal a conserved arginine on FLCN as the catalytic arginine finger and lead us to interpret our structure as an on-pathway intermediate. These data reveal features of a GAP-GTPase interaction and the structure of a critical component of the nutrient-sensing mTORC1 pathway.


Subject(s)
Carrier Proteins/ultrastructure , Cryoelectron Microscopy , Monomeric GTP-Binding Proteins/ultrastructure , Multiprotein Complexes/ultrastructure , Proto-Oncogene Proteins/ultrastructure , Tumor Suppressor Proteins/ultrastructure , Arginine/metabolism , Biocatalysis , Carrier Proteins/chemistry , GTPase-Activating Proteins/metabolism , HEK293 Cells , Humans , Hydrolysis , Models, Molecular , Monomeric GTP-Binding Proteins/chemistry , Multiprotein Complexes/chemistry , Protein Conformation , Protein Multimerization , Proto-Oncogene Proteins/chemistry , Tumor Suppressor Proteins/chemistry
2.
Nat Rev Mol Cell Biol ; 21(4): 246, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32005970

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

3.
Nat Rev Mol Cell Biol ; 21(4): 183-203, 2020 04.
Article in English | MEDLINE | ID: mdl-31937935

ABSTRACT

The mTOR pathway integrates a diverse set of environmental cues, such as growth factor signals and nutritional status, to direct eukaryotic cell growth. Over the past two and a half decades, mapping of the mTOR signalling landscape has revealed that mTOR controls biomass accumulation and metabolism by modulating key cellular processes, including protein synthesis and autophagy. Given the pathway's central role in maintaining cellular and physiological homeostasis, dysregulation of mTOR signalling has been implicated in metabolic disorders, neurodegeneration, cancer and ageing. In this Review, we highlight recent advances in our understanding of the complex regulation of the mTOR pathway and discuss its function in the context of physiology, human disease and pharmacological intervention.


Subject(s)
TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Aging/metabolism , Animals , Autophagy/physiology , Humans , Metabolic Diseases/metabolism , Neoplasms/metabolism , Nutritional Status/physiology , Protein Biosynthesis/physiology , Signal Transduction/physiology
4.
Cell ; 168(6): 960-976, 2017 03 09.
Article in English | MEDLINE | ID: mdl-28283069

ABSTRACT

The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.


Subject(s)
Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Aging/metabolism , Animals , Diabetes Mellitus/metabolism , Glucose/metabolism , Humans , Muscles/metabolism , Neoplasms/metabolism
5.
Cell ; 168(5): 890-903.e15, 2017 02 23.
Article in English | MEDLINE | ID: mdl-28162770

ABSTRACT

The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates of enzymes, and the molecular functions of uncharacterized proteins. Comparisons of differentially essential genes between Ras-dependent and -independent lines uncover synthetic lethal partners of oncogenic Ras. Screens in both human AML and engineered mouse pro-B cells converge on a surprisingly small number of genes in the Ras processing and MAPK pathways and pinpoint PREX1 as an AML-specific activator of MAPK signaling. Our findings suggest general strategies for defining mammalian gene networks and synthetic lethal interactions by exploiting the natural genetic and epigenetic diversity of human cancer cells.


Subject(s)
Gene Regulatory Networks , Leukemia, Myeloid, Acute/genetics , Animals , Carrier Proteins , Cell Line, Tumor , Clustered Regularly Interspaced Short Palindromic Repeats , Epigenesis, Genetic , Genes, Essential , Humans , MAP Kinase Signaling System , Mice , Mitochondrial Proteins , Protein Processing, Post-Translational , ras Proteins/genetics
6.
Cell ; 169(2): 258-272.e17, 2017 Apr 06.
Article in English | MEDLINE | ID: mdl-28388410

ABSTRACT

A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis-an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.


Subject(s)
Culture Media/chemistry , Multienzyme Complexes/antagonists & inhibitors , Orotate Phosphoribosyltransferase/antagonists & inhibitors , Orotidine-5'-Phosphate Decarboxylase/antagonists & inhibitors , Uric Acid/metabolism , Aged , Animals , Cell Culture Techniques , Cell Line, Tumor , Fluorouracil/pharmacology , Glucose/metabolism , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Male , Mice , Middle Aged , Multienzyme Complexes/chemistry , Orotate Phosphoribosyltransferase/chemistry , Orotidine-5'-Phosphate Decarboxylase/chemistry , Protein Domains , Pyrimidines/biosynthesis
7.
Cell ; 171(3): 642-654.e12, 2017 Oct 19.
Article in English | MEDLINE | ID: mdl-29053970

ABSTRACT

The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth.


Subject(s)
Amino Acid Transport Systems/metabolism , Amino Acids, Essential/metabolism , Lysosomes/metabolism , Multiprotein Complexes/metabolism , TOR Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Amino Acid Transport Systems/chemistry , Amino Acid Transport Systems/genetics , Animals , Arginine/metabolism , Cell Line , Cell Line, Tumor , Humans , Male , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Inbred C57BL , Sequence Alignment
8.
Cell ; 166(5): 1324-1337.e11, 2016 Aug 25.
Article in English | MEDLINE | ID: mdl-27565352

ABSTRACT

Mitochondria house metabolic pathways that impact most aspects of cellular physiology. While metabolite profiling by mass spectrometry is widely applied at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. We describe a method for the rapid and specific isolation of mitochondria and use it in tandem with a database of predicted mitochondrial metabolites ("MITObolome") to measure the matrix concentrations of more than 100 metabolites across various states of respiratory chain (RC) function. Disruption of the RC reveals extensive compartmentalization of mitochondrial metabolism and signatures unique to the inhibition of each RC complex. Pyruvate enables the proliferation of RC-deficient cells but has surprisingly limited effects on matrix contents. Interestingly, despite failing to restore matrix NADH/NAD balance, pyruvate does increase aspartate, likely through the exchange of matrix glutamate for cytosolic aspartate. We demonstrate the value of mitochondrial metabolite profiling and describe a strategy applicable to other organelles.


Subject(s)
Metabolic Networks and Pathways , Metabolome , Mitochondria/metabolism , Electron Transport/genetics , HeLa Cells , Humans , Pyruvic Acid/metabolism , Pyruvic Acid/pharmacology
9.
Cell ; 165(1): 153-164, 2016 Mar 24.
Article in English | MEDLINE | ID: mdl-26972053

ABSTRACT

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 µM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway.


Subject(s)
Arginine/metabolism , Carrier Proteins/metabolism , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes/metabolism , Protein Multimerization , TOR Serine-Threonine Kinases/metabolism
10.
Cell ; 161(1): 67-83, 2015 Mar 26.
Article in English | MEDLINE | ID: mdl-25815986

ABSTRACT

For organisms to coordinate their growth and development with nutrient availability, they must be able to sense nutrient levels in their environment. Here, we review select nutrient-sensing mechanisms in a few diverse organisms. We discuss how these mechanisms reflect the nutrient requirements of specific species and how they have adapted to the emergence of multicellularity in eukaryotes.


Subject(s)
Bacteria/metabolism , Signal Transduction , Bacteria/genetics , Biological Evolution , Eukaryota/genetics , Eukaryota/metabolism , Food
11.
Cell ; 162(3): 540-51, 2015 Jul 30.
Article in English | MEDLINE | ID: mdl-26232224

ABSTRACT

The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation.


Subject(s)
Aspartic Acid/biosynthesis , Cell Proliferation , Electron Transport , Mitochondria/metabolism , Aspartate Aminotransferase, Cytoplasmic/metabolism , Aspartic Acid/metabolism , DNA, Mitochondrial/genetics , Humans , Jurkat Cells , Mutation , Phenformin/pharmacology , Pyruvic Acid/metabolism
12.
Cell ; 158(5): 1094-1109, 2014 Aug 28.
Article in English | MEDLINE | ID: mdl-25171410

ABSTRACT

It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the "mesenchymal metabolic signature" (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits.


Subject(s)
Epithelial-Mesenchymal Transition , Pyrimidines/metabolism , Animals , Carcinoma/metabolism , Cell Line, Tumor , Dihydrouracil Dehydrogenase (NADP)/genetics , Flow Cytometry , Gene Expression Profiling , Humans , Mesoderm/cytology , Mesoderm/metabolism , Mice , RNA, Small Interfering/metabolism
13.
Nature ; 607(7919): 610-616, 2022 07.
Article in English | MEDLINE | ID: mdl-35831510

ABSTRACT

Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients1,2. Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively3-5. Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 ß-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through ß-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes6. The scaffold orients the WD40 ß-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway.


Subject(s)
Amino Acids , Cryoelectron Microscopy , Multiprotein Complexes , Nutrients , Protein Subunits , Humans , Amino Acids/metabolism , Arginine , Carrier Proteins , Leucine , Mechanistic Target of Rapamycin Complex 1/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Nutrients/metabolism , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolism , Proteins
14.
Nature ; 608(7921): 209-216, 2022 08.
Article in English | MEDLINE | ID: mdl-35859173

ABSTRACT

Mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple nutrients, including the essential amino acid leucine1. Recent work in cultured mammalian cells established the Sestrins as leucine-binding proteins that inhibit mTORC1 signalling during leucine deprivation2,3, but their role in the organismal response to dietary leucine remains elusive. Here we find that Sestrin-null flies (Sesn-/-) fail to inhibit mTORC1 or activate autophagy after acute leucine starvation and have impaired development and a shortened lifespan on a low-leucine diet. Knock-in flies expressing a leucine-binding-deficient Sestrin mutant (SesnL431E) have reduced, leucine-insensitive mTORC1 activity. Notably, we find that flies can discriminate between food with or without leucine, and preferentially feed and lay progeny on leucine-containing food. This preference depends on Sestrin and its capacity to bind leucine. Leucine regulates mTORC1 activity in glial cells, and knockdown of Sesn in these cells reduces the ability of flies to detect leucine-free food. Thus, nutrient sensing by mTORC1 is necessary for flies not only to adapt to, but also to detect, a diet deficient in an essential nutrient.


Subject(s)
Adaptation, Physiological , Diet , Drosophila Proteins , Drosophila melanogaster , Leucine , Sestrins , Adaptation, Physiological/genetics , Animal Feed , Animals , Autophagy , Diet/veterinary , Drosophila Proteins/deficiency , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Food Preferences , Leucine/deficiency , Leucine/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Neuroglia/metabolism , Sestrins/deficiency , Sestrins/genetics , Sestrins/metabolism , Signal Transduction
15.
Nature ; 609(7929): 1005-1011, 2022 09.
Article in English | MEDLINE | ID: mdl-36131016

ABSTRACT

Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.


Subject(s)
Esters , Glycerophospholipids , Inositol Phosphates , Lysosomes , Membrane Glycoproteins , Molecular Chaperones , Animals , Biomarkers/cerebrospinal fluid , Biomarkers/metabolism , Child , Esters/metabolism , Glycerophospholipids/cerebrospinal fluid , Glycerophospholipids/metabolism , Humans , Inositol Phosphates/cerebrospinal fluid , Inositol Phosphates/metabolism , Lysosomal Storage Diseases/cerebrospinal fluid , Lysosomal Storage Diseases/genetics , Lysosomal Storage Diseases/metabolism , Lysosomes/metabolism , Lysosomes/pathology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Neuronal Ceroid-Lipofuscinoses/cerebrospinal fluid , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/metabolism
16.
Cell ; 149(2): 274-93, 2012 Apr 13.
Article in English | MEDLINE | ID: mdl-22500797

ABSTRACT

The mechanistic target of rapamycin (mTOR) signaling pathway senses and integrates a variety of environmental cues to regulate organismal growth and homeostasis. The pathway regulates many major cellular processes and is implicated in an increasing number of pathological conditions, including cancer, obesity, type 2 diabetes, and neurodegeneration. Here, we review recent advances in our understanding of the mTOR pathway and its role in health, disease, and aging. We further discuss pharmacological approaches to treat human pathologies linked to mTOR deregulation.


Subject(s)
Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Animals , Humans , Metabolic Diseases/metabolism , Neoplasms/metabolism , Neurodegenerative Diseases/metabolism , Stem Cells/metabolism
17.
Cell ; 169(2): 361-371, 2017 04 06.
Article in English | MEDLINE | ID: mdl-28388417
18.
Cell ; 150(6): 1196-208, 2012 Sep 14.
Article in English | MEDLINE | ID: mdl-22980980

ABSTRACT

The mTOR Complex 1 (mTORC1) pathway regulates cell growth in response to numerous cues, including amino acids, which promote mTORC1 translocation to the lysosomal surface, its site of activation. The heterodimeric RagA/B-RagC/D GTPases, the Ragulator complex that tethers the Rags to the lysosome, and the v-ATPase form a signaling system that is necessary for amino acid sensing by mTORC1. Amino acids stimulate the binding of guanosine triphosphate to RagA and RagB but the factors that regulate Rag nucleotide loading are unknown. Here, we identify HBXIP and C7orf59 as two additional Ragulator components that are required for mTORC1 activation by amino acids. The expanded Ragulator has nucleotide exchange activity toward RagA and RagB and interacts with the Rag heterodimers in an amino acid- and v-ATPase-dependent fashion. Thus, we provide mechanistic insight into how mTORC1 senses amino acids by identifying Ragulator as a guanine nucleotide exchange factor (GEF) for the Rag GTPases.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Amino Acids/metabolism , Drosophila/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Proteins/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Sequence , Animals , GTP Phosphohydrolases/metabolism , Guanine Nucleotide Exchange Factors/chemistry , HEK293 Cells , Humans , Mechanistic Target of Rapamycin Complex 1 , Molecular Sequence Data , Multiprotein Complexes , TOR Serine-Threonine Kinases
19.
Proc Natl Acad Sci U S A ; 121(35): e2322755121, 2024 Aug 27.
Article in English | MEDLINE | ID: mdl-39163330

ABSTRACT

The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates cell growth and metabolism in response to many environmental cues, including nutrients. Amino acids signal to mTORC1 by modulating the guanine nucleotide loading states of the heterodimeric Rag GTPases, which bind and recruit mTORC1 to the lysosomal surface, its site of activation. The Rag GTPases are tethered to the lysosome by the Ragulator complex and regulated by the GATOR1, GATOR2, and KICSTOR multiprotein complexes that localize to the lysosomal surface through an unknown mechanism(s). Here, we show that mTORC1 is completely insensitive to amino acids in cells lacking the Rag GTPases or the Ragulator component p18. Moreover, not only are the Rag GTPases and Ragulator required for amino acids to regulate mTORC1, they are also essential for the lysosomal recruitment of the GATOR1, GATOR2, and KICSTOR complexes, which stably associate and traffic to the lysosome as the "GATOR" supercomplex. The nucleotide state of RagA/B controls the lysosomal association of GATOR, in a fashion competitively antagonized by the N terminus of the amino acid transporter SLC38A9. Targeting of Ragulator to the surface of mitochondria is sufficient to relocalize the Rags and GATOR to this organelle, but not to enable the nutrient-regulated recruitment of mTORC1 to mitochondria. Thus, our results reveal that the Rag-Ragulator complex is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway and underscore that mTORC1 activation requires signal transduction on the lysosomal surface.


Subject(s)
Amino Acids , Lysosomes , Mechanistic Target of Rapamycin Complex 1 , Monomeric GTP-Binding Proteins , Nutrients , Signal Transduction , Mechanistic Target of Rapamycin Complex 1/metabolism , Lysosomes/metabolism , Humans , Amino Acids/metabolism , Monomeric GTP-Binding Proteins/metabolism , Nutrients/metabolism , Animals , Mice , Adaptor Proteins, Signal Transducing/metabolism , HEK293 Cells
20.
Immunity ; 46(6): 1045-1058.e6, 2017 06 20.
Article in English | MEDLINE | ID: mdl-28636954

ABSTRACT

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection.


Subject(s)
B-Lymphocytes/physiology , Clonal Selection, Antigen-Mediated , Germinal Center/immunology , Multiprotein Complexes/metabolism , T-Lymphocytes, Helper-Inducer/immunology , TOR Serine-Threonine Kinases/metabolism , Animals , Antibody Affinity , Cell Cycle , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multiprotein Complexes/genetics , Receptors, Antigen, B-Cell/genetics , Sirolimus/pharmacology , Somatic Hypermutation, Immunoglobulin , TOR Serine-Threonine Kinases/genetics
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