Fig. 5: The LAMTOR complex binds PI3P and PI(3,5)P2 through LAMTOR1 and LAMTOR3.
From: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth
a, A schematic showing the LAMTOR complex (LT1–LT5) anchoring RagGTPases at the lysosomal surface and linking Rag–mTORC1 signalling to MTM1-regulated phosphoinositides, PI3P and PI(3,5)P2. b, Representative immunoblots from liposome flotation assays assessing the binding of recombinant LAMTOR complexes to phosphoinositides. Glutathione-S-transferase (GST)–HRS (PI3P binding) served as a positive control. LT1 binds PI3P, whereas LT3 binds PI(3,5)P2 and PI3P, detected in the top fraction (dashed red boxes). Data are from three biologically independent experiments. c, The structural analysis identifying positively charged residues on LT1 and LT3 implicated in phosphoinositide binding. Representative immunoblots from PIP-bead assays using recombinant wild-type or mutant LAMTOR complexes (two biologically independent experiments). LT1(K103/K104) mutations impair PI3P binding, whereas LT3(K6/R7 and K34) mutations impair PI3P and PI(3,5)P2 binding (see also Extended Data Fig. 8e). d, Representative images of 7-day myotubes expressing wild-type or mutant LT1 and LT3. Individual LT1 or LT3 mutations do not alter lysosomal localization, whereas combined LT1/LT3 mutations reduce lysosomal association. Data are shown as mean ± s.d., n = 58 cell areas from three biologically independent experiments. One-way ANOVA with Dunnett’s multiple-comparisons test. Scale bar, 100 µm. e, LT1 or LT3 phosphoinositide-binding mutations reduce mTORC1 activity (p-S6K) in MTM1-KOCas9 myotubes. Data are shown as mean ± s.d. from three biologically independent experiments. One-way ANOVA with Dunnett’s multiple-comparisons test. f, The expression of LT1(K103/K104) or LT3(K6/R7/K34) mutants improves the differentiation of MTM1-KOCas9 myotubes as assessed by fusion index and myotube area. Data are shown as mean ± s.d., n = 34 myotubes per condition from three biologically independent experiments; one-way ANOVA with Dunnett’s multiple-comparisons test. Scale bar, 100 µm. g, Representative immunoblots from pulse–chase assays showing impaired LAMTOR complex turnover in MTM1-KOCas9 myotubes. Data are shown as mean ± s.d. from three biologically independent experiments. h, The co-expression of LT1 and LT3 phosphoinositide-binding mutants restores LAMTOR complex turnover in MTM1-KOCas9 myotubes. Representative immunoblots from three biologically independent experiments. i, Polyubiquitin trap assays showing reduced LT1 and LT3 ubiquitination in MTM1-KOCas9 myotube lysates. MLN4924 (a selective inhibitor of NEDD8-activating enzyme and E3 ubiquitin ligases) was used as a positive control. Representative immunoblots from three biologically independent experiments. For b–e and g–i, protein molecular weight is indicated in kDa. IN, input; B, bottom; M, middle; T, top; EV, empty vector. Illustrations in a and b created in BioRender; Karim, H. https://biorender.com/n6ucxpz (2026).
