Fig 2: DRAM1 Drives LAT1 and SLC1A5 Amino Acid Transporters to Lysosomes, which Export Amino Acids and Induce mTOR Activation(A) Dram1flox/flox MEFs expressing Cre recombinase (-/-) or a control vector (fl/fl) were starved for 3 h in EBSS with or without glutamine before treatment with 0.8 mM leucine in EBSS for 20 min. Intracellular leucine was determined by liquid chromatography-mass spectrometry (LC-MS) analysis.(B) Saos TetOn-DRAM1-myc-his cells overexpressing V5-tagged SLC1A5 were grown for 24 h in the presence or absence of doxycycline (1 µg/mL). Cells were fixed and stained for LAMP2 and SLC1A5(V5-tag). Scale bars represent 50 µm.(C) Quantification of colocalization coefficients of LAMP2 with SLC1A5-V5-tagged in Saos TetOn-DRAM1 cells treated or not with doxycycline for 24 h (average of at least 25 cells).(D) Saos2 TetOn-DRAM1 cells expressing LAMP1-RFP-FLAG2× were induced with or without doxycycline for 24 h. Cells were then starved for 3 h in EBSS prior to 20-min treatment with 0.8 mM methyl-leucine ester. Lysosome-enriched fractions were analyzed by western blot for levels of LAMP2, SLC1A5, and SLC7A5.(E and F) Quantification of 3 independent experiments was determined using ImageJ. Results represent the relative amounts of SLC7A5 (E) or SLC1A5 (F) normalized to LAMP2 and compared to non-induced starved DRAM1 cells. *p < 0.05.(G) Intracellular leucine (Leu) or methyl leucine ester (CH3-Leu) were analyzed as described in (A) in cells that were starved in EBSS for 3 h before treatment with EBSS supplemented with 0.8 mM methyl-leucine ester for 20 min.(H and I) Dram1flox/flox MEF expressing Cre recombinase (-/-) or a control vector (fl/fl) cells were starved in the absence or presence of glutamine for 3 h before treatment with EBSS supplemented with 0.8 mM leucine or 0.8 mM methyl leucine ester (H) or 0.8 mM methyl leucine ester and 10 mM D-phenylalanine (I) as indicated for 20 min. mTOR activation was detected by measuring phospho-S6 kinase levels by western blot. ERK2 was used as a loading control.(J) Amino acid transporters SLC1A5 or SLC7A5 were downregulated using siRNAs in Dram1flox/flox MEFs expressing Cre recombinase (-/-) or a control vector (fl/fl). mTOR activation was detected by measuring phospho-S6 kinase levels by western blot after cells were starved 3 h in EBSS containing glutamine and treated with 0.8 mM methyl leucine ester for 20 min. ERK2 was used as a loading control.(K) Metabolites from lysosomal fractions were analyzed for intralysosomal leucine content using high-performance liquid chromatography (HPLC)-MS. Results of 3 independent experiments are presented as the amount of lysosomal leucine measured for each condition and normalized to non-induced DRAM1 cells treated with methyl-leucine ester. *p < 0.05.For micrographs, scale bars represent 50 µm. (A and G) Data are mean ± SD. (C–F and K) Data are mean ± SEM.See also Figure S4.