Editor | XiThe decision of whether a cell initiates autophagy is crucial. When nutrients are abundant, cells reduce catabolism by inhibiting the synthesis of lysosomes. Lysosomes are organelles responsible for the degradation and recycling of cellular components. The formation of lysosomes is regulated by the transcription factor TFEB. When cells have sufficient nutrients and do not require more lysosomes, TFEB is inhibited through phosphorylation by mTORC1.The mTORC1 complex is a hub for sensing the extracellular environment, promoting cell growth while inhibiting catabolism. In nutrient-rich environments, mTORC1 is recruited to the lysosomal surface and activated by the Rag GTPases-Ragulator complex, phosphorylating downstream substrates to promote various signaling pathways. mTORC1 has over 50 substrates, and previous studies on phosphorylation mechanisms mainly focused on S6K kinase and 4E-BP1. However, increasing evidence suggests that the phosphorylation of TFEB by mTORC1 differs from that of S6K and 4E-BP1.First, TFEB is recruited to mTORC1 by Rag GTPases dimers and does not contain the TOS sequence present in S6K and 4E-BP1. Secondly, the phosphorylation of TFEB is influenced only by the levels of amino acids in the extracellular environment, rather than by growth factors. Therefore, the molecular mechanisms by which TFEB is phosphorylated by mTORC1 may also differ from those of S6K and 4E-BP1.On January 25, 2023, a team led by James H. Hurley (with Dr. Cui Zhicheng as the first author) from the University of California, Berkeley, in collaboration with Andrea Ballabio from Italy, published an article in Nature titled Structure of the lysosomal mTORC1–TFEB–Rag–Ragulator megacomplex, revealing the atypical phosphorylation mechanism of TFEB by mTORC1.
The authors reconstituted all subunits involved in the phosphorylation of TFEB in vitro and then analyzed their three-dimensional structure using cryo-electron microscopy. The key to successful in vitro reconstitution was co-expressing TFEB and Rag GTPases, along with mutations to stabilize the interaction between TFEB and Rag.Surprisingly, the final structure showed that the phosphorylation of TFEB by mTORC1 requires additional Rag-Ragulator. The authors referred to the previously observed copy as the “typical” Rag-Ragulator and the new, previously unreported copy as the “atypical” Rag-Ragulator.The “atypical” Rag-Ragulator plays a crucial role in the phosphorylation of TFEB. First, the “atypical” Rag binds to TFEB, revealing the mechanism by which TFEB is recruited to mTORC1. Then, the Lamtor1 subunit in the “atypical” Ragulator interacts with the “typical” RagC subunit to ensure the stability of TFEB during recruitment. The authors subsequently tested the effects of many key interaction mutations on the phosphorylation of TFEB.Due to the large number of unstructured regions in TFEB, only about a quarter of the structure was resolved. However, since the active site of mTORC1 is only about 30 angstroms from the last resolved amino acid of TFEB at position 108, the authors inferred that the phosphorylation sites S122 and S142 of TFEB would be precisely phosphorylated. However, the article also notes that it is still unknown how the site S211 is precisely phosphorylated.In summary, the latest three-dimensional structure shows that the molecular mechanism of TFEB phosphorylation by mTORC1 is unique compared to S6K and 4EBP1. This work provides structural evidence for atypical mTORC1 signaling, allowing selective control of TFEB activity under specific conditions. Enhancing TFEB activation may help treat lysosomal storage diseases, promote the clearance of toxic aggregates in neurons, and prevent non-alcoholic fatty liver through lipid clearance. The structures revealed in this article identify several key interactions that can be utilized for these purposes.Original link:https://doi.org/10.1038/s41586-022-05652-7
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