Fig. 4

Figure 4. Defective DENND5A triggers lysosomal degradation of melanosomal cargoes and fails to associate with SNX1-decorated vesicles. A. Representative confocal microscopy images of control MNT-1 (siCON, upper) and after silencing DENND5A (siDENND5A, lower). LAMP1 (green) and PMEL (PMEL-C antibody, red) labeled MNT-1 cells are labeled and shown in merged channels, with higher magnification on the right-hand side. Scale bars: 10 μm, 1 μm for the magnified panels. B. Colocalization measured using images from 2 independent experiments. C. Western blotting and (D) quantification of melanosome markers for control (siCON) treated MNT-1 and siDENND5A treated MNT-1 cells after treatment of Golgi-stop and lysosome protease inhibitor leupeptin, respectively. E. DENND5A^KO 1 MNT-1 cells transiently introduced with V5-DENND5A^WT (upper) and V5-DENND5A^mt (lower). Transfected cells stained for DENND5A (green) and SNX1 (red). Scale bars: 10 μm, 1 μm for the magnified panels. F. Colocalization coefficiency measured using images from 2 independent experiments. Data quantification is presented as mean ± SEM. D. Two-tailed, Unpaired t test. B., F. Mann-Whitney test. G. Immunoprecipitation of V5-tag in DENND5 ^KO 1 cells overexpressing the wild-type (V5-DENND5A^WT) and truncated (V5-DENND5A^mt) constructs. H. Proposed working model showing the homeostasis of melanosome maturation and lysosomal degradation coordinated by DENND5A-SNX1 interaction (left). C-del DENND5A loses its interaction with SNX1 and triggers missorting of PMEL from en route to melanosomes to degradation subdomain (stage I melanosome),²⁶ resulting in lysosomal entry and degradation of PMEL (right). IB, immunoblotting; IP, immunoprecipitation; RPT, repeat domain. ∗P <.05; ∗∗P <.01.