Fig. 1. Liver FLCN selectively promotes mTORC1-mediated cytoplasmic sequestration of TFE3, without affecting canonical mTORC1 signaling.

(A and B) Model of selective mTORC1 regulation. P, phosphorylated; PRAS40, proline-rich AKT substrate of 40 kDa; DEPTOR, DEP domain-containing mTOR-interacting protein; mLST8, target of rapamycin complex subunit LST8. (C) Schematic of FLCN liver deletion to yield control and hepatocyte-specific Flcn-null (LiFKO) mice. (D) FLCN protein expression in livers of control and LiFKO mice fed normal chow and euthanized after overnight fast followed by 4 hours of refeeding. HSP90, heat shock protein 90. (E) TFE3 protein expression in the indicated subcellular fractions from livers of control, LiFKO, and Tfe3 knockout mice fed normal chow and euthanized ad lib. S.E., short exposure; L.E., long exposure; H3, histone H3. (F) TFE3 protein expression in the nucleus from livers of control (Con.) and Raptor liver-KO mice, euthanized after overnight fasting and 4 hours of refeeding. See fig. S1A for whole cell and cytoplasmic fractions. p:total, fraction of phosphorylated protein to total protein. (G and H) Phosphorylation of mTORC1 targets in control and LiFKO mice fed either (G) normal chow or (H) 7 or 8 days of FPC diet (TD190142) and sugar water, and euthanized after overnight fast and ~4 hours of refeeding. pS6 240/4, phospho-ribosomal protein S6 (Ser^240/244); S6, ribosomal protein S6; p4E-BP1 S65, phospho-4E-BP1 (Ser⁶⁵); p4E-BP1 Th37, phospho-4E-BP1 (Thr³⁷). (I) Phosphorylation of Lipin1, assayed by immunoprecipitation (IP) with anti-Lipin1 followed by immunoblotting for phospho-Lipin1, in liver lysates of control and LiFKO mice fed 9 days of FPC diet (TD190142) and sugar water and euthanized at 10 p.m. ad lib. IgG, immunoglobulin G. (J) TFE3 protein expression in the indicated subcellular fractions from control and liver Tsc1 KO mice fed normal chow and euthanized ad lib. *P < 0.05, **P < 0.01; Student’s two-tailed t test. Data are depicted as mean ± SEM.