Ragulator and SLC38A9 activate the Rag GTPases through noncanonical GEF mechanisms

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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Reduced expression of C/EBPβ-LIP extends health- and lifespan in mice

10.1101/250100 ◽

2018 ◽

Author(s):

Christine Müller ◽

Laura M. Zidek ◽

Tobias Ackermann ◽

Tristan de Jong ◽

Peng Liu ◽

...

Keyword(s):

Metabolic Disorders ◽

Upstream Open Reading Frame ◽

Reading Frame ◽

Physical Decline ◽

Genetic Ablation ◽

Mechanistic Target Of Rapamycin ◽

Age Related ◽

Extended Lifespan ◽

Mtorc1 Pathway ◽

Factor C

AbstractAgeing is associated with physical decline and the development of age-related diseases such as metabolic disorders and cancer. Few conditions are known that attenuate the adverse effects of ageing, including calorie restriction (CR) and reduced signalling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Synthesis of the metabolic transcription factor C/EBP β ‐LIP is stimulated by mTORC1, which critically depends on a short upstream open reading frame (uORF) in the C/EBP β-mRNA. Here we describe that reduced C/EBP β ‐LIP expression due to genetic ablation of the uORF delays the development of age-associated phenotypes in mice. Moreover, female C/EBP βΔuORF mice display an extended lifespan. Since LIP levels increase upon aging in wt mice, our data reveal an important role for C/EBPβ in the aging process and suggest that restriction of LIP expression sustains health and fitness. Thus, therapeutic strategies targeting C/EBP β ‐LIP may offer new possibilities to treat age-related diseases and to prolong healthspan.

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Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor

Journal of Virology ◽

10.1128/jvi.01221-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 4

Author(s):

Vânia Passos ◽

Thomas Zillinger ◽

Nicoletta Casartelli ◽

Amelie S. Wachs ◽

Shuting Xu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Subcellular Localization ◽

Transmembrane Protein ◽

Type I ◽

Accessory Gene ◽

Protein Levels ◽

Specificity And Sensitivity ◽

Hiv 1

ABSTRACT When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface. IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.

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Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.12.8985140 ◽

1996 ◽

Vol 44 (12) ◽

pp. 1481-1488 ◽

Cited By ~ 50

Author(s):

V A Tanner ◽

T Ploug ◽

J H Tao-Cheng

Keyword(s):

Subcellular Localization ◽

Pc12 Cells ◽

Cell Cultures ◽

Transmembrane Protein ◽

Secretory Granules ◽

Secretory Vesicle ◽

Secretory Vesicles ◽

Experimental Conditions ◽

Gold Probe ◽

20 Nm

We demonstrated the subcellular localization of SV2, a transmembrane protein associated with neuroendocrine secretory vesicles, in NGF-treated PC12 cells by preembedding EM immunocytochemistry (ICC), using a small gold probe followed by silver enhancement. The use of a multiwell chamber slide substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types of secretory vesicles was compared quantitatively to that of another secretory vesicle-associated transmembrane protein, synaptophysin. In cultures under similar experimental conditions, the ratio of SV2 vs synaptophysin ICC staining on synaptic vesicle dusters was about 1:1, whereas it was about 9:1 on LDCV membranes. Furthermore, whereas SV2 is localized on the membranes of the LDCVs, chromogranin A, an acidic protein in secretory granules, is clearly in the core of the LDCVs. This is the first demonstration of these two antigens in such dose (approximately 20 nm) yet distinct compartments within a single organelle.

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ENZYMIC ACTIVITY AND CONTROL AS RELATED TO SUBCELLULAR LOCALIZATION

Journal of Food Science ◽

10.1111/j.1365-2621.1972.tb02684.x ◽

1972 ◽

Vol 37 (4) ◽

pp. 524-529 ◽

Cited By ~ 4

Author(s):

H. O. HULTIN

Keyword(s):

Subcellular Localization ◽

Enzymic Activity ◽

And Control

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Assesment of Endometrial Vascularization with Immunohistochemical CD34 Assay in Recurrent Pregnancy Loss: A Case Control Study

Gynecology Obstetrics and Reproductive Medicine ◽

10.21613/gorm.2016.664 ◽

2017 ◽

pp. 1

Author(s):

Emine Aydın ◽

Taner Usta

Keyword(s):

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Case Control Study ◽

Transmembrane Protein ◽

Vessel Diameter ◽

Control Group ◽

Control Groups ◽

Significant Difference ◽

And Control ◽

Control Study

Objectives: We compared the endometrial vascularization in hysteroscopic endometrial samplings between recurrent pregnancy loss (RPL) and control group.Study Design: We prospectively evaluated hysteroscopic endometrial samplings from RPL and control groups. CD34 transmembrane protein was used for evaluating endometrial vascularization. The vascularization was assessed based on thickness of vessels, diameter of the largest vessel, and number of vessels per mm2 in CD34-stained slides.Results: There was no significant difference in demographic findings and vascularization, such as largest vessel diameter (p: 0.572), and number of vessels per mm2 (p: 0.982) between the two groups.Conclusion: The cycling endometrium is a highly angiogenic tissue and may play a role in the etiology of RPL. However, we find a weak relationship between endometrial vascularization and RPL.

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AGIA Tag System for Ultrastructural Protein Localization Analysis in Blood-Stage Plasmodium falciparum

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.777291 ◽

2021 ◽

Vol 11 ◽

Author(s):

Masayuki Morita ◽

Bernard N. Kanoi ◽

Naoaki Shinzawa ◽

Rie Kubota ◽

Hiroyuki Takeda ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Subcellular Localization ◽

Western Blotting ◽

Protein Localization ◽

Transmembrane Protein ◽

Physiological Role ◽

Immunofluorescence Assay ◽

Blood Stage ◽

Parasite Development ◽

High Signal

Precise subcellular localization of proteins is the key to elucidating the physiological role of these molecules in malaria parasite development, understanding of pathogenesis, and protective immunity. In Plasmodium falciparum, however, detection of proteins in the blood-stage parasites is greatly hampered by the lack of versatile protein tags which can intrinsically label such molecules. Thus, in this study, to develop a novel system that can be used to evaluate subcellular localization of known and novel proteins, we assessed the application of AGIA tag, consisting of 9 amino acids (EEAAGIARP), in P. falciparum blood-stage parasites. Specifically, AGIA-tagged ring-infected erythrocyte surface antigen (RESA-AGIA) was episomally expressed in P. falciparum 3D7 strain. The RESA-AGIA protein was detected by Western blotting and immunofluorescence assay (IFA) using recombinant rabbit anti-AGIA tag monoclonal antibody (mAb) with a high signal/noise ratio. Similarly, AGIA-tagged multidrug resistance protein 1 (MDR1-AGIA), as an example of polyptic transmembrane protein, was endogenously expressed and detected by Western blotting and IFA with anti-AGIA tag mAb. Immunoelectron microscopy of the RESA-AGIA transfected merozoites revealed that mouse anti-RESA and the rabbit anti-AGIA mAb signals could definitively co-localize to the dense granules. Put together, this study demonstrates AGIA tag/anti-AGIA rabbit mAb system as a potentially useful tool for elucidating the subcellular localization of new and understudied proteins in blood-stage malaria parasites at the nanometer-level resolution.

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Location-dependent maintenance of intrinsic susceptibility to mTORC1-driven tumorigenesis

Life Science Alliance ◽

10.26508/lsa.201800218 ◽

2019 ◽

Vol 2 (2) ◽

pp. e201800218 ◽

Cited By ~ 5

Author(s):

Gabrielle V Rushing ◽

Asa A Brockman ◽

Madelyn K Bollig ◽

Nalin Leelatian ◽

Bret C Mobley ◽

...

Keyword(s):

Signal Transduction ◽

Brain Tumors ◽

Subventricular Zone ◽

Tumor Development ◽

Direct Link ◽

Mechanistic Target Of Rapamycin ◽

Cells Of Origin ◽

Growth Properties ◽

Neural Stem Progenitor Cells ◽

Mtorc1 Pathway

Neural stem/progenitor cells (NSPCs) of the ventricular–subventricular zone (V-SVZ) are candidate cells of origin for many brain tumors. However, whether NSPCs in different locations within the V-SVZ differ in susceptibility to tumorigenic mutations is unknown. Here, single-cell measurements of signal transduction intermediates in the mechanistic target of rapamycin complex 1 (mTORC1) pathway reveal that ventral NSPCs have higher levels of signaling than dorsal NSPCs. These features are linked with differences in mTORC1-driven disease severity: introduction of a pathognomonic Tsc2 mutation only results in formation of tumor-like masses from the ventral V-SVZ. We propose a direct link between location-dependent intrinsic growth properties imbued by mTORC1 and predisposition to tumor development.

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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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Perspective: The Potential Role of Essential Amino Acids and the Mechanistic Target of Rapamycin Complex 1 (mTORC1) Pathway in the Pathogenesis of Child Stunting

Advances in Nutrition ◽

10.3945/an.116.013276 ◽

2016 ◽

Vol 7 (5) ◽

pp. 853-865 ◽

Cited By ~ 19

Author(s):

Richard D Semba ◽

Indi Trehan ◽

Marta Gonzalez-Freire ◽

Klaus Kraemer ◽

Ruin Moaddel ◽

...

Keyword(s):

Amino Acids ◽

Potential Role ◽

Essential Amino Acids ◽

Target Of Rapamycin ◽

Child Stunting ◽

Mechanistic Target Of Rapamycin ◽

Mtorc1 Pathway

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Intracellular signals direct integrin localization to sites of function in embryonic muscles.

The Journal of Cell Biology ◽

10.1083/jcb.134.1.217 ◽

1996 ◽

Vol 134 (1) ◽

pp. 217-226 ◽

Cited By ~ 25

Author(s):

M D Martin-Bermudo ◽

N H Brown

Keyword(s):

Subcellular Localization ◽

Transmembrane Protein ◽

Drosophila Embryo ◽

Cellular Polarity ◽

Intracellular Signals ◽

Tendon Cells ◽

Segment Polarity ◽

Set Up ◽

Longitudinal Muscles

In the Drosophila embryo, the alphaPS2betaPS integrin heterodimer is localized tightly at the termini of the multinucleate muscles where they attach to the alphaPS1betaPS-containing epidermal tendon cells. Here we examine the basis for alphaPS2betaPS integrin subcellular localization. We show that the betaPS cytoplasmic tail is sufficient to direct the localization of a heterologous transmembrane protein, CD2, to the muscle termini in vivo. This localization does not occur via an association with structures set up by the endogenous betaPS integrins, since it can occur even in the absence of the betaPS protein. Furthermore, the subcellular localization of the alphaPS2betaPS integrin is not dependent on any other interactions between the muscles and the tendon cells. In embryos that lack the segmental tendon cells, due to a mutation removing the related segment polarity genes engrailed and invected, alphaPS2betaPS is still localized to the muscle termini even though the ventral longitudinal muscles are not attached to the epidermis, but instead are attached end to end. Thus the alphaPS2betaPS integrin can be localized by an intracellular mechanism within the muscles. Our results challenge the view that the transmission of signals from the extracellular environment via integrins is required for the organization of the cytoskeleton and the resultant cellular polarity.

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