Fig. 3: Blockage of proper lysosomal enzyme sorting and delivery disconnects mTORC1 localization on lysosomes and activity towards cytoplasmic substrates.
From: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources
a, A schematic model of lysosomal enzyme sorting at the Golgi and delivery to lysosomes that depends on the GNPTAB enzyme. b,c, Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells using confocal microscopy (b) and its quantification (c). Cells were transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated as indicated. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. The composition of all media is described in Methods. n = 44–50 individual cells from five independent fields per condition (see also Methods). d, Immunoblots with lysates from HEK293FT WT cells transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions as described in b, probed with the indicated antibodies. e,f, Functional characterization of GNPTAB KO HEK293FT cells. Two independent KO clones show impaired cathepsin D (CTSD) processing and mannose-6-phosphate (M6P)-tagging of proteins. Note also the differential effect of GNPTAB loss on the different mTORC1 substrates (TFEB de-phosphorylated in KO cells, whereas S6K phosphorylation is largely unaffected) (e). The pro-form of CTSD is aberrantly secreted in the medium of GNPTAB KO cells (f). g,h, Lysosomal accumulations of mTOR are lost in GNPTAB KOs (g) and quantification of mTOR/LAMP2 colocalization (h). n = 50 individual cells from five independent fields per condition. For microscopy, magnified insets are shown to the right. Scale bars, 25 μm and for insets, 5 μm. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. **P < 0.01, ****P < 0.0001. Source numerical data and unprocessed blots are available in Source data.
