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. 2018 May 10;7:e36620. doi: 10.7554/eLife.36620

Figure 4. SLC37A3 and ATRAID transport N-BPs from the lumen of lysosomes into the cytosol.

(A) Live imaging of HEK 293 T cells that express Halo tagged SLC37A3 (SLC37A3-Halo) and have internalized AlexaFlour 647 labeled zoledronate (AF647-ZLN). SLC37A3-Halo is labeled with Janelia flour 549 (JF549). SLC37A3-Halo is expressed at a lower-than-endogenous level and has been verified to be functional (data not shown). The scale bar represents 10 µm. The image displayed is a representative example chosen from five similar images. (B) Radioactive uptake assay measuring total intracellular radioactivity in indicated HEK 293 T cells treated with 3H-alendronate. Data depict mean and s.d. for biological triplicate measurements. (C) Radioactive uptake assay measuring levels of radioactivity in subcellular fractions in indicated HEK 293 T cells treated with 3H-alendronate. Data depict mean with s.d. for biological duplicate measurements. Significance was determined using unpaired two-way ANOVA test. Effect of genotype: F(2,6) = 74.93, p<0.0001; effect of subcellular location: F(1,6) = 864.9, p<0.0001; effect of interaction between genotype and subcellular location: F(2,6) = 312.4, p<0.0001. (D) Radioactive uptake assay measuring levels of radioactivity in lysosomes purified from indicated HEK 293 T cells treated with 3H-alendronate. Data depict mean and s.d. for biological triplicate measurements. Significance was determined using two-tailed unpaired t-test with equal s.d. Comparison between wild-type and ATRAIDKO cells: df = 4, t = 36.24, p<0.0001. Comparison between wild-type and SLC37A3KO cells: df = 4, t = 17.96, p<0.0001. HEK 293 T cells were treated with 1 μCi/mL 3H-alendronate for 24 hr in (B–C) and 3 hr in (D). ****: p<0.0001.

Figure 4.

Figure 4—figure supplement 1. Quality controls for the radioactive uptake assays.

Figure 4—figure supplement 1.

(A) Immunoblot against markers for cytosol (GAPDH), plasma membrane (Caveolin1), early endosome (EEA1), lysosome (LAMP1), nucleosol (RaN-BP3), and nucleoskeleton (LaminB1) in cytosolic fractions and membranous fractions in wild-type, ATRAIDKO and SLC37A3KO HEK 293 T cells, demonstrating successful subcellular fractionation in the fractionation-based radioactive uptake assay. Cyt: cytosolic fraction. Mem: membranous fraction. Tot: total cell lysate. (B–D) Localization of HA tagged TMEM192 (TMEM192-HA), a lysosomal protein we expressed in wild-type (B), ATRAIDKO (C) and SLC37A3KO (D) HEK 293 T cells and used as a handle to immuno-precipitate lysosomes, shown with a lysosomal marker, LAMP2, demonstrating correct localization of TMEM192-HA to lysosomes and, consequently, successful purification of lysosomes in the lysosome-purification-based uptake assay. Scale bars represent 10 µm. Each image displayed is the representative example chosen from at least five similar images.