Figure 3. SLC37A3 and ATRAID form a lysosomal complex and are inter-dependent for their stable expression.

(A–D) Localization of HA-tagged SLC37A3 (SLC37A3-HA) (A–B) and V5-tagged short isoform of ATRAID (sATRAID-V5) (C–D) shown with markers for lysosomes (LAMP2, (A and C) and the plasma membrane (Na⁺/K⁺-ATPase, (B and D). (E) Co-localization of SLC37A3-HA and sATRAID-V5. Nuclei were stained with DAPI in blue. Scale bars represent 10 µm. Each image displayed is the representative example chosen from at least five similar images. (F) Reciprocal co-IP of SLC37A3A3-HA and sATRAID-V5 in KO² HEK 293 T cells stably overexpressing both proteins. In each negative control cell line, one of the two tagged proteins was replaced with GFP tagged with the same epitope. (G) Immunoblots measuring SLC37A3-HA protein levels in various cells, showing that deletion of ATRAID significantly reduces the protein level of SLC37A3-HA. The un-glycosylated population of SLC37A3 that appears in the absence of ATRAID is marked with an asterisk. (H) Immunoblots measuring ATRAID-V5 protein levels in various cells, demonstrating that deletion of SLC37A3 significantly reduces the protein level of ATRAID-V5. IP, immunoprecipitation. IB, immunoblot. KO²: ATRAID^KO; SLC37A3^KO.

Figure 3—figure supplement 1. Additional evidence supporting that SLC37A3 and ATRAID form a lysosomal complex.

(A) mRNA levels of ATRAID and SLC37A3 in various HEK 293 T cells used in this study. Data depict mean and s.d. for technical triplicate measurements. (B) Dose responses to alendronate in KO² HEK 293 T cells complemented with either only SLC37A3-HA or both ATRAID-V5 and SLC37A3-HA, compared with those in wild-type and KO² HEK 293 T cells. Data depict mean and s.d. for biological triplicate measurements. (C–H) Localization of SLC37A3-HA (C), sATRAID-V5 (D) or lATRAID-V5 (F–H) shown with LAMP2 (F), EEA1 (C, D and H) and Na⁺/K⁺-ATPase (G), and co-localization of SLC37A3-HA and lATRAID-V5 (E). Scale bars represent 10 µm. Each image displayed is the representative example chosen from at least five similar images. Note that in E), there exists a subpopulation of lATRAID-V5 that localizes to the plasma membrane but not with SLC37A3-HA. Such distribution is likely a result of a higher-than-endogenous expression level of lATRAID-V5 in KO² +SLC37A3-HA + lATRAID-V5 HEK 293 T cells (A), as such distribution is not observed in ATRAID^KO +lATRAID-V5 HEK 293 T cells (G), which express lATRAID-V5 at a lower-than-endogenous level (A). KO²: ATRAID^KO; SLC37A3^KO. sATRAID: short isoform of ATRAID. lATRAID: long isoform of ATRAID.

Figure 3—figure supplement 2. Additional evidence supporting that SLC37A3 and ATRAID depend on each other for stable expression.

(A–C) Analysis of IF images comparing the expression levels of SLC37A3-HA (A) or both isoforms of ATRAID-V5 (B and C) in their respective single knockout backgrounds and the KO² background. The two images in each sub-figure were acquired with the same setting and adjusted to the same contrast. In each image a background area (turquoise or blue, inside nuclei, where no stain should be present) and a signal area (orange or red) were selected and the distribution of pixel values within each area was plotted in a histogram. The outlines in the histogram are color-coded to match the boarders of selected areas. (D–E) Polysome profiling experiment assessing the translation efficiency of SLC37A3 transcripts in SLC37A3^KO and KO² backgrounds. Lysates from indicated cell lines were analyzed on a gradient station and fractionated into five fractions: untranslated transcripts (UT), small and large ribosome subunits (SL), lowly translated transcripts (LT), medially translated transcripts (MT) and highly translated transcripts (HT). The level of SLC37A3 transcripts relative to the level of TBP (TATA-binding protein) transcripts in each fraction was measured and plotted. The SL fraction was excluded from the analysis. A total RNA fraction was included as a reference. No overall shift was observed in the distribution of SLC37A3 transcripts in the KO² background compared to that in the SLC37A3^KO background, suggesting that the translation efficiency of SLC37A3 is not affected by the absence of ATRAID. (F) Immunoblot comparing the glycosylation patterns of SLC37A3 in SLC37A3^KO and KO² backgrounds. Lysates from SLC37A3^KO + SLC37A3-HA (lane 1–3) and KO² + SLC37A3-HA (lane 4–6) HEK 293 T cells were left untreated (lane 1 and 4), treated with PNGase-F (lane 2 and 5), or with Endo H (lane 3 and 6). The band corresponding to an un-glycosylated population of SLC37A3 that is present in the absence of ATRAID but not in the presence of ATRAID is marked with an asterisk. (G) Immunoblot comparing the glycosylation patterns of total cellular SLC37A3 and the sub-population of SLC37A3 that interacts with ATRAID. In KO² HEK 293 T cells over-expressing SLC37A3-HA and sATRAID-V5, proteins that interact with sATRAID-V5 were purified with immuno-precipitation against V5 epitope and compared with total proteins in the lysate. The pre-IP total lysate (lane 1–2) and anti-V5 IP eluate (lane 3–4) were either left untreated (lane 1 and 3) or treated with PNGase F (lane 2 and 4) and analyzed by blotting against SLC37A3-HA. IF: immunofluorescence. IB: immunoblot. KO²: ATRAID^KO; SLC37A3^KO. PNGase F: peptide: N-glycosidase F, an enzyme that removes all asparagine (N)-linked sugar modifications from glycoproteins. Endo H: endoglycosidase H, an enzyme that only removes high mannose sugar moieties on ER glycoproteins that have not been processed by the Golgi apparatus.