Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex

The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagAGDP:RagCGTP GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo–electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.

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Structural mechanism for amino acid-dependent Rag GTPase switching by SLC38A9

10.1101/2020.07.28.225524 ◽

2020 ◽

Author(s):

Simon A. Fromm ◽

Rosalie E. Lawrence ◽

James H. Hurley

Keyword(s):

Amino Acid ◽

Cytoplasmic Tail ◽

Stable States ◽

Gtp Hydrolysis ◽

Interacting Protein ◽

Structural Mechanism ◽

Cryo Electron Microscopy ◽

Amino Acid Levels ◽

Outstanding Question ◽

Rag Gtpase

The mechanistic target of rapamycin complex 1 (mTORC1) couples cell growth to nutrient, energy and growth factor availability (1–3). mTORC1 is activated at the lysosomal membrane when amino acids are replete via the Rag guanosine triphosphatases (GTPases) (4–6). Rags exist in two stable states, an inactive (RagA/BGDP:RagC/DGTP) and active (RagA/BGTP:RagC/DGDP) state, during low and high cellular amino acid levels (4, 5). The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator (7, 8), and the FLCN:FNIP (FLCN-interacting protein) GTPase activating protein (GAP) complex (9), and prevents activation of the Rag dimer during amino acid starvation (10, 11). How the LFC is released upon amino acid refeeding is a major outstanding question in amino-acid dependent Rag activation. Here we show that the cytoplasmic tail of the lysosomal solute carrier family 38 member 9 (SLC38A9), a known Rag activator (12–14), destabilizes the LFC. By breaking up the LFC, SLC38A9 triggers the GAP activity of FLCN:FNIP toward RagC. We present the cryo electron microscopy (cryo-EM) structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre and post GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.

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The GATOR–Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids

Science ◽

10.1126/science.aaz0863 ◽

2020 ◽

Vol 370 (6514) ◽

pp. 351-356

Author(s):

Geoffrey G. Hesketh ◽

Fotini Papazotos ◽

Judy Pawling ◽

Dushyandi Rajendran ◽

James D. R. Knight ◽

...

Keyword(s):

Amino Acids ◽

Cell Growth ◽

Target Of Rapamycin ◽

Lysosomal Degradation ◽

Oncogenic Ras ◽

Mechanistic Target Of Rapamycin ◽

Independent Mechanism ◽

Guanosine Triphosphatase ◽

Rag Gtpase ◽

Mtorc1 Activation

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR–Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase–independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.

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Structural basis for the docking of mTORC1 on the lysosomal surface

Science ◽

10.1126/science.aay0166 ◽

2019 ◽

Vol 366 (6464) ◽

pp. 468-475 ◽

Cited By ~ 37

Author(s):

Kacper B. Rogala ◽

Xin Gu ◽

Jibril F. Kedir ◽

Monther Abu-Remaileh ◽

Laura F. Bianchi ◽

...

Keyword(s):

Electron Microscopy ◽

Growth Factors ◽

Protein Kinase ◽

Nucleotide Binding ◽

Target Of Rapamycin ◽

Structural Basis ◽

Mechanistic Target Of Rapamycin ◽

Rag Gtpases ◽

Cryo Electron Microscopy ◽

Guanosine Triphosphatase

The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)–Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP–RagC/D•GDP) permits mTORC1 association. We used cryo–electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor “claw” threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome.

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TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism

Nature Communications ◽

10.1038/s41467-021-24499-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nicola Alesi ◽

Elie W. Akl ◽

Damir Khabibullin ◽

Heng-Jia Liu ◽

Anna S. Nidhiry ◽

...

Keyword(s):

Transcription Factor ◽

Tuberous Sclerosis Complex ◽

Renal Tumors ◽

Gtpase Activating Protein ◽

Mechanistic Target Of Rapamycin ◽

Lysosome Biogenesis ◽

Pulmonary Lymphangioleiomyomatosis ◽

Transcription Factor Eb ◽

Rag Gtpase ◽

Dependent Mechanism

AbstractTuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/− mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.

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The lysosomal Rag-Ragulator complex licenses RIPK1– and caspase-8–mediated pyroptosis by Yersinia

Science ◽

10.1126/science.abg0269 ◽

2021 ◽

Vol 372 (6549) ◽

pp. eabg0269

Author(s):

Zengzhang Zheng ◽

Wanyan Deng ◽

Yang Bai ◽

Rui Miao ◽

Shenglin Mei ◽

...

Keyword(s):

Cell Death ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Host Cells ◽

Caspase 8 ◽

Death Domain ◽

Interacting Protein ◽

A Genome ◽

Infection Inhibition ◽

Guanosine Triphosphatase

Host cells initiate cell death programs to limit pathogen infection. Inhibition of transforming growth factor–β–activated kinase 1 (TAK1) by pathogenic Yersinia in macrophages triggers receptor-interacting serine-threonine protein kinase 1 (RIPK1)–dependent caspase-8 cleavage of gasdermin D (GSDMD) and inflammatory cell death (pyroptosis). A genome-wide CRISPR screen to uncover mediators of caspase-8–dependent pyroptosis identified an unexpected role of the lysosomal folliculin (FLCN)–folliculin-interacting protein 2 (FNIP2)–Rag-Ragulator supercomplex, which regulates metabolic signaling and the mechanistic target of rapamycin complex 1 (mTORC1). In response to Yersinia infection, Fas-associated death domain (FADD), RIPK1, and caspase-8 were recruited to Rag-Ragulator, causing RIPK1 phosphorylation and caspase-8 activation. Pyroptosis activation depended on Rag guanosine triphosphatase activity and lysosomal tethering of Rag-Ragulator but not mTORC1. Thus, the lysosomal metabolic regulator Rag-Ragulator instructs the inflammatory response to Yersinia.

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Tyrosine phosphorylation of guanosine triphosphatase activating protein by activation via surface IgG in human B cells

Blood ◽

10.1182/blood.v81.6.1535.bloodjournal8161535 ◽

1993 ◽

Vol 81 (6) ◽

pp. 1535-1539

Author(s):

H Yagura ◽

N Oyaizu ◽

S Pahwa

Keyword(s):

B Cells ◽

Tyrosine Phosphorylation ◽

Signal Transduction Pathway ◽

Dependent Manner ◽

Transduction Pathway ◽

Gtpase Activating Protein ◽

Human B Cells ◽

Associated Proteins ◽

Guanosine Triphosphatase ◽

Dose Dependent

In this study, we analyzed tyrosine phosphorylation of guanosine triphosphatase (GTPase) activating protein in human B cells stimulated through surface IgG, using Western blot and immunoprecipitation. Stimulation through surface IgG induced the tyrosine phosphorylation of GTPase-activating protein (GAP) and two associated proteins, a 190-Kd protein and a 62-Kd protein, within 1 minute and in a dose-dependent manner. This tyrosine phosphorylation was blocked by Genistein (Extrasynthese, Genay, France). These data suggest that GTPase- activating protein is involved in a signal transduction pathway initiated from surface IgG in human B cells.

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EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

The Journal of Cell Biology ◽

10.1083/jcb.201508086 ◽

2016 ◽

Vol 212 (3) ◽

pp. 297-306 ◽

Cited By ~ 21

Author(s):

Atsuhiro Nakajo ◽

Shin-ichiro Yoshimura ◽

Hiroko Togawa ◽

Masataka Kunii ◽

Tomohiko Iwano ◽

...

Keyword(s):

Small Intestine ◽

Plasma Membrane ◽

Membrane Proteins ◽

Basolateral Membrane ◽

Membrane Curvature ◽

Apical Plasma Membrane ◽

Interacting Protein ◽

Guanosine Triphosphatase ◽

Biochemical Analyses ◽

Interacting Protein Complex

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain–binding protein 1–like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1–dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

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Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association

Science Advances ◽

10.1126/sciadv.1501502 ◽

2016 ◽

Vol 2 (3) ◽

pp. e1501502 ◽

Cited By ~ 36

Author(s):

Thiemo Sprink ◽

David J. F. Ramrath ◽

Hiroshi Yamamoto ◽

Kaori Yamamoto ◽

Justus Loerke ◽

...

Keyword(s):

Translation Initiation ◽

Protein Biosynthesis ◽

Initiation Factor ◽

Structural Basis ◽

Initiation Complex ◽

Cryo Electron Microscopy ◽

Initiation Factor 2 ◽

High Resolution Structure ◽

Guanosine Triphosphatase ◽

Resolution Structure

Throughout the four phases of protein biosynthesis—initiation, elongation, termination, and recycling—the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAiMet in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation.

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Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801287115 ◽

2018 ◽

Vol 115 (23) ◽

pp. E5279-E5288 ◽

Cited By ~ 24

Author(s):

Minji Lee ◽

Jong Hyun Kim ◽

Ina Yoon ◽

Chulho Lee ◽

Mohammad Fallahi Sichani ◽

...

Keyword(s):

Protein Synthesis ◽

Trna Synthetase ◽

Gtp Hydrolysis ◽

Mtorc1 Signaling ◽

Amino Acid Sensing ◽

Gtpase Cycle ◽

Rag Gtpase ◽

Cancer Tissues ◽

Mtorc1 Activation ◽

Hydrolysis Of

A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating “ON” switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an “OFF” switch by controlling GTP hydrolysis of RagB in the Rag GTPase–mTORC1 axis. The LRS–RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP–GDP cycle of the RagD–RagB pair, rather than the RagC–RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD–RagB pair can overcome the absence of the RagC–RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD–RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.

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Ragulator and SLC38A9 activate the Rag GTPases through noncanonical GEF mechanisms

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811727115 ◽

2018 ◽

Vol 115 (38) ◽

pp. 9545-9550 ◽

Cited By ~ 44

Author(s):

Kuang Shen ◽

David M. Sabatini

Keyword(s):

Subcellular Localization ◽

Transmembrane Protein ◽

Mechanistic Target Of Rapamycin ◽

Rag Gtpases ◽

Locking Mechanism ◽

Mtorc1 Pathway ◽

Loaded State ◽

Rag Gtpase ◽

And Control ◽

Growth Pathway

The mechanistic target of rapamycin complex 1 (mTORC1) growth pathway detects nutrients through a variety of sensors and regulators that converge on the Rag GTPases, which form heterodimers consisting of RagA or RagB tightly bound to RagC or RagD and control the subcellular localization of mTORC1. The Rag heterodimer uses a unique “locking” mechanism to stabilize its active (GTPRagA–RagCGDP) or inactive (GDPRagA–RagCGTP) nucleotide states. The Ragulator complex tethers the Rag heterodimer to the lysosomal surface, and the SLC38A9 transmembrane protein is a lysosomal arginine sensor that upon activation stimulates mTORC1 activity through the Rag GTPases. How Ragulator and SLC38A9 control the nucleotide loading state of the Rag GTPases remains incompletely understood. Here we find that Ragulator and SLC38A9 are each unique guanine exchange factors (GEFs) that collectively push the Rag GTPases toward the active state. Ragulator triggers GTP release from RagC, thus resolving the locked inactivated state of the Rag GTPases. Upon arginine binding, SLC38A9 converts RagA from the GDP- to the GTP-loaded state, and therefore activates the Rag GTPase heterodimer. Altogether, Ragulator and SLC38A9 act on the Rag GTPases to activate the mTORC1 pathway in response to nutrient sufficiency.

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