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. 2019 Oct 3;76(1):163–176.e8. doi: 10.1016/j.molcel.2019.07.021

Figure 4.

Figure 4

DRAM1 Interacts with SCAMP3 to Drive SLC1A5 and LAT1 to Lysosome Membranes

(A) 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) and then treated for 4 h with cycloheximide (100 μg/mL) or anisomycin (10 μg/mL). Cells were fixed in 4% paraformaldehyde and stained for Lamp2 and V5.

(B) Saos2 TetOn-DRAM1-myc-his cells overexpressing V5-tagged SLC1A5 were, where indicated, treated with doxycycline for 24 h and then treated for 4 h with cycloheximide (100 μg/mL) or anisomycin (10 μg/mL). SLC1A5 expression was detected using an anti-V5 antibody: a short and long exposure is shown. DRAM-1 levels were detected using an anti-Myc tag antibody. Actin was used as a loading control

(C) Saos2 TetOn-DRAM-1 cells treated with or without doxycycline for 24 h were lysed prior to immunoprecipitation of DRAM-1 using anti-Myc-tag antibody. Immunoblotting from total protein extracts (INPUT) or elutions (IP DRAM-1) was undertaken to detect SCAMP3, DRAM-1 (Myc tag), and ERK2.

(D) Saos Tet-On-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 in 4% paraformaldehyde and stained for SCAMP3, DRAM1 (Myc-tag), and SLC1A5 (V5-tag).

(E) SCAMP3 was downregulated using siRNA in Saos-2 TetOn-DRAM1-myc-his cells overexpressing V5-tagged SLC1A5, grown for 24 h in the presence or absence of doxycycline (1 μg/mL). Cells were fixed in 4% paraformaldehyde and stained for SLC1A5(V5-tag) and LAMP2.

(F) Colocalization coefficients were determined using Zeiss Zen Black software (average of at least 25 cells). Data are mean ± SEM.

(G) SCAMP3 was downregulated using siRNA in Saos TetOn-DRAM1-myc-his cells overexpressing V5-tagged SLC1A5, then, where indicated, treated with Dox for 24 h, and then starved for 3 h in EBSS before treatment with EBSS containing leucine for 20 min. mTORC1 activation was evaluated by measuring phospho-S6 kinase and phospho-S6 levels by western blot. Levels of total S6 kinase, S6, and actin were used as loading controls. DRAM1 expression was detected using an anti-Myc-tag antibody, and SCAMP3 knockdown was measured using an anti- SCAMP3 antibody.