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The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels 1 . Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B–GATOR2–mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer. SAR1B, which is conserved between mammals and nematodes, is a leucine sensor that is involved in regulating mTORC1 signalling and potentially has a role in the development of lung cancer.

The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo–electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.

The mechanistic target of rapamycin complex 1 (mTORC1) plays a central role in regulating cell growth and metabolism by responding to cellular nutrient conditions. The activity of mTORC1 is controlled by Rag GTPases, which are anchored to lysosomes via Ragulator, a pentameric protein complex consisting of membrane-anchored p18/LAMTOR1 and two roadblock heterodimers. Here we report the crystal structure of Ragulator in complex with the roadblock domains of RagA-C, which helps to elucidate the molecular basis for the regulation of Rag GTPases. In the structure, p18 wraps around the three pairs of roadblock heterodimers to tandemly assemble them onto lysosomes. Cellular and in vitro analyses further demonstrate that p18 is required for Ragulator-Rag GTPase assembly and amino acid-dependent activation of mTORC1. These results establish p18 as a critical organizing scaffold for the Ragulator-Rag GTPase complex, which may provide a platform for nutrient sensing on lysosomes. mTORC1 activity is controlled through Rag GTPases, which are anchored to the lysosome through the Ragulator. Here, the authors give molecular insights into Ragulator-Rag GTPase assembly and present the crystal structures of the Ragulator alone and in complex with the RagA-C roadblock domains.

Rice seed storage proteins glutelin and α-globulin are synthesized in the endoplasmic reticulum (ER) and deposited in protein storage vacuoles (PSVs). Sar1, a small GTPase, acts as a molecular switch to regulate the assembly of coat protein complex II, which exports secretory protein from the ER to the Golgi apparatus. To reveal the route by which glutelin and α-globulin exit the ER, four putative Sar1 genes (OsSar1a/b/c/d) were cloned from rice, and transgenic rice were generated with Sar1 overexpressed or suppressed by RNA interference (RNAi) specifically in the endosperm under the control of the rice glutelin promoter. Overexpression or suppression of any OsSar1 did not alter the phenotype. However, simultaneous knockdown of OsSar1a/b/c resulted in floury and shrunken seeds, with an increased level of glutelin precursor and decreased level of the mature α- and β-subunit. OsSar1abc RNAi endosperm generated numerous, spherical, novel protein bodies with highly electron-dense matrixes containing both glutelin and α-globulin. Notably, the novel protein bodies were surrounded by ribosomes, showing that they were derived from the ER. Some of the ER-derived dense protein bodies were attached to a blebbing structure containing prolamin. These results indicated that OsSar1a/b/c play a crucial role in storage proteins exiting from the ER, with functional redundancy in rice endosperm, and glutelin and α-globulin transported together from the ER to the Golgi apparatus by a pathway mediated by coat protein complex II.

SAMHD1 restricts the infection of dendritic cells by human immunodeficiency virus type 1. Margottin-Goguet and colleagues show that SAMHD1 limits viral DNA synthesis by hydrolyzing intracellular dNTPs. SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)–HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.

Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex—comprising DEPDC5, NPRL2 and NPRL3—regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1–Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1–Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2–NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase. Cryo-electron microscopy and biochemical analyses of the GATOR1 protein complex reveal that two binding modes underpin its ability to regulate Rag GTPases as a GTPase-activating protein for RAGA. Restraining the RAGA GAP mTORC1 is a master growth regulator that senses nutrient signals such as amino acids and glucose to coordinate many cellular processes. mTORC1 is regulated by the Rag GTPases, which are in turn regulated by several protein complexes including GATOR1, which serves as a GTPase-activating protein (GAP) for RAGA. Here, David Sabatini and colleagues use cryo-electron microscropy to determine the structures of GATOR1 and of GATOR1 bound to the Rag GTPases, and perform biochemical experiments to investigate GAP function. They find that GATOR1 adopts an extended architecture with the NPRL2 subunit linking the two other subunits DEPDC5 and NPRL3. Unexpectedly, the GATOR1–Rag GTPases structure represents an inhibitory state, where an interaction of DEPDC5 with RAGA inhibits GATOR1 GAP activity. Instead, a weaker interaction of the NPRL2–NPRL3 heterodimer with RAGA executes GAP function. Such an inhibitory mechanism has not been seen before for other GAPs.

How macrophages avoid HIV-1 infection HIV-1 is unable to replicate efficiently in dendritic cells, the antigen-presenting tissue cells that function in both innate and adaptive immunity. Other primate lentiviruses, including HIV-2 and some simian immunodeficiency viruses, express a protein called Vpx that is able to overcome the block to replication. Two groups now report the identification of the restriction factor in dendritic cells and macrophages that is overcome by Vpx. Vpx is found to induce degradation of the protein SAMHD1. Mutations in SAMHD1 cause Aicardi–Goutières syndrome, a disorder characterized by inappropriate activation of the immune system. Knockdown of SAMHD1 increases HIV-1 replication in dendritic cells, which could be important for generating appropriate immune responses to the virus. The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection 1 , 2 , 3 , 4 , 5 , 6 . Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi–Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response 7 . We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.

Background Human SAMHD1 is a triphosphohydrolase that restricts the replication of retroviruses, retroelements and DNA viruses in noncycling cells. While modes of action have been extensively described for human SAMHD1, only little is known about the regulation of SAMHD1 in the mouse. Here, we characterize the antiviral activity of murine SAMHD1 with the help of knockout mice to shed light on the regulation and the mechanism of the SAMHD1 restriction and to validate the SAMHD1 knockout mouse model for the use in future infectivity studies. Results We found that endogenous mouse SAMHD1 restricts not only HIV-1 but also MLV reporter virus infection at the level of reverse transcription in primary myeloid cells. Similar to the human protein, the antiviral activity of murine SAMHD1 is regulated through phosphorylation at threonine 603 and is limited to nondividing cells. Comparing the susceptibility to infection with intracellular dNTP levels and SAMHD1 phosphorylation in different cell types shows that both functions are important determinants of the antiviral activity of murine SAMHD1. In contrast, we found the proposed RNase activity of SAMHD1 to be less important and could not detect any effect of mouse or human SAMHD1 on the level of incoming viral RNA. Conclusion Our findings show that SAMHD1 in the mouse blocks retroviral infection at the level of reverse transcription and is regulated through cell cycle-dependent phosphorylation. We show that the antiviral restriction mediated by murine SAMHD1 is mechanistically similar to what is known for the human protein, making the SAMHD1 knockout mouse model a valuable tool to characterize the influence of SAMHD1 on the replication of different viruses in vivo.

SAMHD1 has emerged as a novel HIV restriction factor that inhibits viral replication by limiting dNTP availability. Here, Schwartz and colleagues discuss the studies that led to the identification and characterization of SAMHD1, and speculate on why HIV-1 does not encode the SAMHD1-targeting protein, Vpx. HIV replication is limited by cellular restriction factors, such as APOBEC and tetherin, which themselves are counteracted by viral proteins. SAMHD1 was recently identified as a novel HIV restriction factor in myeloid cells, and was shown to be blocked by the lentiviral protein Vpx. SAMHD1 limits viral replication through an original mechanism: it hydrolyses intracellular dNTPs in non-cycling cells, thus decreasing the amount of these key substrates, which are required for viral DNA synthesis. In this Progress article, we describe how SAMHD1 regulates the pool of intracellular nucleotides to control HIV replication and the innate immune response.

Antiretroviral role for SAMHD1 protein Mutations in SAMHD1 protein are associated with the human autoimmune disease Aicardi–Goutières syndrome, and SAMHD1 was recently shown to be responsible for restriction of HIV-1 replication in myeloid cells. Ian Taylor and colleagues reveal a previously unknown function of SAMHD1 that could explain its antivirus role. They provide a crystal structure of the catalytic core of SAMHD1 and show that it is a dGTP-stimulated triphosphohydrolase that hydrolyses dNTPs, the building blocks of DNA. This activity may prevent reverse transcription and viral synthesis of complementary DNA by keeping the concentration of cellular dNTPs at a low level. SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1 ), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells 2 , 3 and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction 4 , 5 , 6 , 7 . SAMHD1 is also associated with Aicardi–Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α 8 . The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.