Fig. 6

FBXO9-mediated ATP6V1A ubiquitination leading to cytoplasmic localization facilitated by HSPA8 A Interaction between HSPA8 and ATP6V1A assessed by co-IP assay. HEK293T cells were co-transfected with indicated constructs. After 36 h, co-IP and immunoblot was performed to detect the associated HA-ATP6V1A using anti-HA antibodies. B Mapping the binding domain of ATP6V1A with HSPA8. Different versions of HA-tagged ATP6V1A, including full-length (FL) and truncated constructs, were co-transfected with FLAG-HSPA8 in HEK293T cells. Co-IP was performed to assess the interaction, and the presence of bound FLAG-HSPA8 was detected using anti-FLAG antibodies. C, D Mapping the binding domain of HSPA8 with ATP6V1A. Different forms of FLAG-tagged HSPA8 constructs, including full-length (FL) and truncated versions, were designed and shown (C). HEK293T cells were co-transfected with HA-ATP6V1A and the corresponding FLAG-tagged HSPA8 constructs. Co-IP assay was performed using anti-FLAG beads to assess the interaction between HSPA8 and ATP6V1A. The presence of bound HA-ATP6V1A was detected using anti-HA antibodies (D). E Impact of FBXO9 knockdown on the interaction between ATP6V1A and HSPA8. HEK293T cells pre-treated with FBXO9-shRNA were co-transfected with HA-ATP6V1A and FLAG-HSPA8. After 36 h, a co-IP assay was performed to evaluate the effect of FBXO9 depletion on the interaction between ATP6V1A and HSPA8. F Effect of FBXO9 knockout on the interaction between endogenous ATP6V1A and HSPA8. FBXO9-knockout H1299 was used and a co-IP assay was performed using anti-ATP6V1A antibody. The presence of the bound endogenous HSPA8 was detected using anti-HSPA8 antibodies. G Role of FBXO9-mediated ubiquitination in modulating the interaction between ATP6V1A and HSPA8. HEK293T cells were co-transfected with FLAG-HSPA8 and either HA-ATP6V1A or HA-ATP6V1A-K393R. The co-IP assay was then conducted to evaluate the interaction between HSPA8 and ATP6V1A. H, I Impact of HSPA8 depletion on lysosomal acidity in A549 cells. HSPA8 was knocked down using shRNA (H, upper). The cells were stained with LysoTracker Red and fluorescence was measured (H, lower). Staining intensity was quantified as outlined in Fig. 5C (I). The scale bar is 50 µM; (P < 0.0001). J, K Impact of HSPA8 depletion on V-ATPase assembly in A549 cells. HSPA8-knockdown cells were fractionated, and immunoblotting was performed using antibodies against ATP6V1A and ATP6VoD (J). Membrane and cytosolic proteins were assessed with LAMP1 and GAPDH as loading controls, respectively. Levels of assembled ATP6V1A were measured and normalized to ATP6VoD in the membrane fraction (K). Results are from three independent experiments (P < 0.0001). L, M Blocking the interaction between HSPA8 and ATP6V1A enhances lysosomal acidity in A549 cells. Cells expressing the A8-40 aa peptide were stained with LysoTracker Red and fluorescence was observed (L). The average staining intensity was quantified as outlined in Fig. 5C. Scale bar = 100 µM. ****P < 0.0001. N, O Blocking the interaction between HSPA8 and ATP6V1A promotes V-ATPase assembly. A549 cells expressing the A8-40 aa peptide were fractionated. Immunoblot analysis was then performed to assess the level of V-ATPase in the cells (P < 0.05), as described in Fig. 6J and K