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. 2023 Aug 10;25(9):1303–1318. doi: 10.1038/s41556-023-01198-6

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — a,b, Mal-CoA, and to a lesser extent acetyl-CoA (Ac-CoA), inhibits TORC1 activity in vitro. TORC1 purified from yeast was used in IVK assays with recombinant Lst4^Loop and co-purified Tco89 proteins as substrates in the presence of increasing concentrations of Mal-CoA, Ac-CoA or CoA. a, Substrate phosphorylation was detected using autoradiography (³²P). Total protein was detected by SYPRO Ruby staining. b, Calculated levels of TORC1 activity (Lst4^Loop phosphorylation; n = 3 independent experiments). c–f, mTORC1 purified from HEK293FT cells was used in IVK assays as in a with recombinant 4E-BP1 protein as the substrate in the presence of increasing concentrations of Mal-CoA (c), Ac-CoA (d) or CoA (e). c–e, Phosphorylation of 4E-BP1 was detected by immunoblotting. f, Levels of mTORC1 activity (n_Mal-CoA = 9, n_Ac-CoA = 6 and n_CoA = 4 independent experiments). g,h, Mal-CoA inhibits TORC1 in an ATP-competitive manner. Increasing ATP concentrations with or without Mal-CoA were used in IVK assays performed as in a. h, Levels of TORC1 activity (Lst4^Loop phosphorylation; n = 3 independent experiments). i,j, Increasing ATP concentrations in the presence or absence of 2 mM Mal-CoA were used in IVK assays performed as in c. j, Levels of mTORC1 activity (n = 4 independent experiments). k, Model of mTOR inhibition by Mal-CoA. When the FA biosynthesis machinery is active, ACC1 converts Ac-CoA to Mal-CoA, which is in turn rapidly converted to palmitate by FASN (left). In contrast, when ACC1 is hyperactive or FASN is downregulated, accumulating Mal-CoA competes with ATP for binding to proximal mTOR molecules, causing their inactivation. Hence, by complexing with ACC1 and FASN, mTORC1 functions as a direct sensor for Mal-CoA to adjust growth and coordinate cellular metabolic activity in response to decreased cellular FA biosynthesis capacity (right). b,f,h,j, Data are the mean ± s.e.m. Ctrl, control; N.C., not calculated. Source numerical data and unprocessed blots are provided.

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