Isolation of Lysosome-Enriched Fractions

Isolation of lysosomes was conducted as described previously (Eguchi et al., 2018 (link); Komori et al., 2023 (link)). Cells on a 10-cm dish were cultured in DMEM containing 1 mM HEPES-NaOH (pH 7.2) and 10% dextran-coated magnetite (DexoMAG 40; Liquids Research Ltd.) for 24 h, and then chased in normal media for 24 h. Cells were harvested with trypsin, centrifuged at 60 × g for 5 min, washed with ice-cold PBS, lysed in 2 ml of ice-cold Buffer A (1 mM HEPES, 15 mM KCl, 1.5 mM Mg(Ac)₂, 1 mM DTT, protease/phosphatase inhibitors) with a Dounce homogenizer, and passed through a 23G needle for eight times. After homogenization, 500 μl of ice-cold Buffer B (220 mM HEPES, 375 mM KCl, 22.5 mM Mg(Ac)₂, 1 mM DTT, 0.1 mM sucrose, and 50 μg/ml DNase I) was immediately added, and samples were inverted for five times, incubated for 5 min, and then centrifuged at 400 × g for 10 min. The supernatant was then applied to an MS Column (Miltenyi Biotec) set on an OctoMACS separator (Miltenyi Biotec) and equilibrated with 0.5% BSA in PBS and the flow through was collected. 1 ml of DNase I solution (50 μg/ml DNase I, 0.1 mM sucrose in PBS) was added and the column was incubated for 10 min and washed with 1 ml of ice-cold sucrose buffer (0.1 mM sucrose in PBS). After removing the column from the OctoMACS separator, lysosomes were eluted with 250 μl of ice-cold sucrose buffer using a plunger.