Fig 2: PP2A catalytic C subunit α (PP2A-Cα) and the scaffold A subunit α/β (PP2A-Aα/β) partially, but constantly, localize to lysosomes, regardless of intracellular AAs or Ins levels.(A, B) Immunofluorescence analysis of HEK293 cells transfected with control siRNA or siRNAs specific for PP2A-Aα/β (A) or PP2A-Cα (B). PP2A-Aα/β (A) and PP2A-Cα (B) (green) were stained with each specific antibody. LAMP1, red; DAPI, blue. Scale bars, 10 μm. Line scans of PP2A-Aα/β (A) and PP2A-Cα (B) (green) together with those of LAMP1 (red) are shown at the bottom. (C, D) Representative immunofluorescence images of p18Rev (C) and p18-KO (D) cells treated with AA and Ins, alone or in combination for 15 min. The areas in the small squares are enlarged and shown in the lower panels. TSC2, green; LAMP1, red; and PP2A-Cα, blue. Scale bars, 10 μm. Line scans of TSC2 (green), LAMP1 (red), and PP2A-Cα (blue) are shown at the bottom of the figure. (C, D, E, F) TSC2-LAMP1 (E) and PP2A-Cα-LAMP1 (F) colocalizations in (C, D) were measured based on Pearson’s correlation coefficient. The mean values of Pearson’s correlation coefficient from six cells and the SEM are shown. P-values were assessed using one-way ANOVA with Tukey’s multiple comparison test. NS, not significant.Source data are available for this figure.

Fig 3: Artificially forced lysosomal TSC2 localization attenuates the phosphorylation levels of T1462.(A, B) LAMP1-Flag-TSC2 (WT or T1395A) was transiently expressed in HEK293 cells. The cells were stimulated with AA and Ins as indicated for 15 min. (A, B) Cell lysates were probed with the indicated antibodies (A, B). (C) Immunofluorescence analysis of LAMP1-Flag-TSC2 transiently expressing HEK293 cells that were AA-starved for 1 h, and subsequently treated with AA and Ins, as indicated, for 15 min. Flag, green; LAMP1, red; DAPI, blue. Scale bars, 10 μm.Source data are available for this figure.

Fig 4: Comprehensive identification of proteins on the lysosomal surface using a proximity-labeling method (Lyso-BioID).(A) Schematic representation of proximity-dependent biotin identification of lysosomal surface proteins (Lyso-BioID) using LAMP1-AGIA-AirID. (B) Representative immunofluorescence images of LAMP1-AGIA-AirID stably expressing HEK293 cells treated with growth medium (GM) (AA(+)) or AA-free medium (AA(−)), alone or in combination with 50 μM biotin for 10 h. AGIA, green; LAMP1, red; streptavidin (SA), blue. Scale bars, 10 μm. (C) LAMP1-AGIA-AirID stably expressing HEK293 cells were cultured in GM or AA(−) medium together with 50 μM biotin for 10 h. Protein biotinylation in LAMP1-AGIA-AirID stably expressing HEK293 cells was confirmed by immunoblotting using an anti-AGIA antibody and SA-HRP. Note that LAMP1 (417 aa, ∼45 kD) is heavily glycosylated at the N-terminal luminal side of the lysosome inner leaflet, providing protection from proteolysis. Therefore, immunoblotting bands for (glycosylated-)LAMP1 appear at higher molecular weight (∼100 kD) than expected. (D) Biotinylated peptides derived from LAMP1-AGIA-AirID stably expressing HEK293 cells cultured in GM or AA(−) medium were detected by LC-MS/MS. Blue dots indicate proteins known to localize on the lysosomal surface. Red dots represent Ser/Thr phosphatases such as PP2A, PP1, calcineurin, and PP2C. (E) Table listing the phosphatase-binding motif sequences and their positions in TSC2 (1,807 aa). TSC2 contains eight PP2A-B56 binding motifs ([L/M/F/I]-x-x-[I/L/V]-x-E-x), one PP1-binding motif ([R/K]-x-V-x-[F/W]), and two calcineurin-binding motifs (L-x-V-P) in TSC2. (F) Cryo-EM structure of the human TSC1-TSC2-TBC1D7 complex (PDB ID: 7DL2), showing a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7, as depicted in Fig 3B. L, M, I, V, and E residues in the PP2A-B56–binding motif sites (273–284; 358–369; 385–396; 442–453; 490–501; 707–718; 1,575–1,586) of TSC2 in (E) are shown as red spheres. The PP2A-B56–binding motif (58–69), PP1-binding motif (1,286–1,297), and calcineurin-binding motifs (1,138–1,149 and 1,249–1,260) are not resolved because of missing structural information. The L361 residue mutated to Pro (L361P), reported in tuberous sclerosis, and the E366 and E1583 residues mutated to Lys (E366K and E1583K) in tumors are indicated by light blue arrows.Source data are available for this figure.

Fig 5: Ins-induced phosphorylation of TSC2 at T1462 is substantially inhibited under AA-free conditions.Phosphorylation levels of AKT at T308 and TSC2 at T1462 monitored in p18Rev and p18-KO MEFs subjected to AA deprivation and subsequent Ins treatment, alone or in combination with AA. Cells were cultured in minimal medium without AA for 1 h and then incubated with medium containing Ins (10 μg/ml) alone or in combination with AA for the indicated time. Cell lysates were analyzed by Western blotting using the indicated antibodies. Note that trial-1 bands of P-TSC2 (P-T1462) and P-AKT (P-T308) are the same ones in Fig 6E.Source data are available for this figure.