Aster-C coordinates with COP I vesicles to regulate lysosomal trafficking and activation of mTORC1.
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ABSTRACT:
SUBMITTER: Zhang J
PROVIDER: S-EPMC7507422 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectiveCholesterol plays a pivotal role in mitochondrial steroidogenesis, membrane structure, and respiration. Mitochondrial membranes are intrinsically low in cholesterol content and therefore must be replenished with cholesterol from other subcellular membranes. However, the molecular mechanisms underlying mitochondrial cholesterol transport remains poorly understood. The Aster-B gene encodes a cholesterol binding protein recently implicated in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (ER). In this study, we investigated the function and underlying mechanism of Aster-B in mediating mitochondrial cholesterol transport.MethodsCRISPR/Cas9 gene editing was carried out to generate cell lines deficient in Aster-B expression. The effect of Aster-B deficiency on mitochondrial cholesterol transport was examined by both confocal imaging analysis and biochemical assays. Deletion mutational analysis was also carried out to identify the function of a putative mitochondrial targeting sequence (MTS) at the N-terminus of Aster-B for its role in targeting Aster-B to mitochondria and in mediating mitochondrial cholesterol trafficking.ResultsAblation of Aster-B impaired cholesterol transport from the ER to mitochondria, leading to a significant decrease in mitochondrial cholesterol content. Aster-B is also required for mitochondrial transport of fatty acids derived from hydrolysis of cholesterol esters. A putative MTS at the N-terminus of Aster-B mediates the mitochondrial cholesterol uptake. Deletion of the MTS or ablation of Arf1 GTPase which is required for mitochondrial translocation of ER proteins prevented mitochondrial cholesterol transport, leading to mitochondrial dysfunction.ConclusionsWe identified Aster-B as a key regulator of cholesterol transport from the ER to mitochondria. Aster-B also coordinates mitochondrial cholesterol trafficking with uptake of fatty acids derived from cholesterol esters, implicating the Aster-B protein as a novel regulator of steroidogenesis.
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Project description:In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.
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Project description:Multiple mitochondrial quality control pathways exist to maintain the health of mitochondria and ensure cell homeostasis. Here, we investigate the role of the endosomal adaptor Tollip during the mitochondrial stress response and identify its interaction and colocalisation with the Parkinson's disease-associated E3 ubiquitin ligase Parkin. The interaction between Tollip and Parkin is dependent on the ubiquitin-binding CUE domain of Tollip, but independent of Tom1 and mitophagy. Interestingly, this interaction is independent of Parkin mitochondrial recruitment and ligase activity but requires an intact ubiquitin-like (UBL) domain. Importantly, Tollip regulates Parkin-dependent endosomal trafficking of a discrete subset of mitochondrial-derived vesicles (MDVs) to facilitate delivery to lysosomes. Retromer function and an interaction with Tom1 allow Tollip to facilitate late endosome/lysosome trafficking in response to mitochondrial stress. We find that upregulation of TOM20-positive MDVs upon mitochondrial stress requires Tollip interaction with ubiquitin, endosomal membranes and Tom1 to ensure their trafficking to the lysosomes. Thus, we conclude that Tollip, via an association with Parkin, is an essential coordinator to sort damaged mitochondrial-derived cargo to the lysosomes.
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Project description:The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid-deficient conditions is not completely understood. Here, we identify ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature-sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.
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Project description:Multiple mitochondrial quality control pathways exist to maintain the health of mitochondria and ensure cell homeostasis. Here we investigate the role of the endosomal adaptor Tollip during mitochondrial stress response and identify its interaction and colocalisation with the Parkinson's disease associated E3 ubiquitin ligase Parkin. The interaction between Tollip and Parkin is dependent on the ubiquitin-binding CUE domain of Tollip, but independent of Tom1 and mitophagy. Interestingly, this interaction is independent of Parkin mitochondrial recruitment and ligase activity but requires an intact Ubiquitin-like (UBL) domain. Importantly, Tollip regulates Parkin-dependent endocytic trafficking of a discrete subset of mitochondria-derived vesicles to facilitate their lysosomal disposal. Retromer function and binding to Tom1 allow Tollip to facilitate late endosome/lysosome trafficking in response to mitochondrial stress. We find that upregulation of TOM20-positive MDVs upon mitochondrial stress requires Tollip interaction with ubiquitin, endosomal membranes, and Tom1 to ensure their trafficking to the lysosomes. Thus, we conclude that Tollip, via an association with Parkin, is an essential coordinator to sort damaged mitochondrial-derived cargo to the lysosomes.
| S-SCDT-EMBOJ-2019-102539 | biostudies-other
Project description:The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant pathways that regulate tumour growth and metastasis. Therefore, we explored the potential crosstalk between these two functionally relevant pathways to coordinate their tumour growth-control functions. We found that a Hippo pathway-related long noncoding RNA, HPR, directly interacts with Raptor, an essential component of mTORC1, to upregulate mTORC1 activation by impairing the phosphorylation of Raptor by AMPK. Knockdown or knockout of HPR in breast cancer and cholangiocarcinoma cells led to a reduction in tumour growth. Compared with HPR WT cells, HPR-overexpressing cells exhibited nuclear accumulation of YAP1, and significantly blocked the downregulation of mTORC1 signalling induced by energy stress. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways in the control of tumour growth.
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Project description:The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid deficient conditions is not completely understood. Here, we identify ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes, are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.
| S-SCDT-EMBOJ-2020-106412 | biostudies-other
Project description:A complex molecular machinery converges on the surface of lysosomes to ensure that the growth-promoting signaling mediated by mechanistic target of rapamycin complex 1 (mTORC1) is tightly controlled by the availability of nutrients and growth factors. The final step in this activation process is dependent on Rheb, a small GTPase that binds to mTOR and allosterically activates its kinase activity. Here we review the mechanisms that determine the subcellular localization of Rheb (and the closely related RhebL1 protein) as well as the significance of these mechanisms for controlling mTORC1 activation. In particular, we explore how the relatively weak membrane interactions conferred by C-terminal farnesylation are critical for the ability of Rheb to activate mTORC1. In addition to supporting transient membrane interactions, Rheb C-terminal farnesylation also supports an interaction between Rheb and the δ subunit of phosphodiesterase 6 (PDEδ). This interaction provides a potential mechanism for targeting Rheb to membranes that contain Arl2, a small GTPase that triggers the release of prenylated proteins from PDEδ. The minimal membrane targeting conferred by C-terminal farnesylation of Rheb and RhebL1 distinguishes them from other members of the Ras superfamily that possess additional membrane interaction motifs that work with farnesylation for enrichment on the specific subcellular membranes where they engage key effectors. Finally, we highlight diversity in Rheb membrane targeting mechanisms as well as the potential for alternative mTORC1 activation mechanisms across species.
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Project description:Using cryo-electron microscopy and molecular characterization, David Sabatini and colleagues provide crucial new insights that validate and expand their model of how amino acids are sensed and signal at the lysosome to activate mechanistic target of rapamycin complex 1 (mTORC1) and cell growth-regulating processes. This work also reveals new therapeutic opportunities for mTORC1-driven diseases.
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Project description:Sensing nutrient availability is essential for appropriate cellular growth, and mTORC1 is a major regulator of this process. Mechanisms causing mTORC1 activation are, however, complex and diverse. We report here an additional important step in the activation of mTORC1, which regulates the efflux of amino acids from lysosomes into the cytoplasm. This process requires DRAM-1, which binds the membrane carrier protein SCAMP3 and the amino acid transporters SLC1A5 and LAT1, directing them to lysosomes and permitting efficient mTORC1 activation. Consequently, we show that loss of DRAM-1 also impacts pathways regulated by mTORC1, including insulin signaling, glycemic balance, and adipocyte differentiation. Interestingly, although DRAM-1 can promote autophagy, this effect on mTORC1 is autophagy independent, and autophagy only becomes important for mTORC1 activation when DRAM-1 is deleted. These findings provide important insights into mTORC1 activation and highlight the importance of DRAM-1 in growth control, metabolic homeostasis, and differentiation.
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