Fig. 2: PDZD8-GFP interacts with GTP-Rab7. | Nature Communications

Fig. 2: PDZD8-GFP interacts with GTP-Rab7.

From: PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria

Fig. 2

a PDZD8 co-localizes with late endosomes. Representative images of U2OS cells expressing PDZD8-GFP and mCherry-Rab7 (late endosomes), mCherry-Rab5 (early endosomes), or LAMP1-mCherry (lysosomes). Scale bar: 10 μm. b PDZD8-GFP is enriched in ER subdomains associated with late endosomes. U2OS cells expressing PDZD8-GFP (green), mCherry-Rab7 (red), and BFP-Sec61β (blue). Arrowheads indicate areas where the BFP-Sec61β-labeled ER co-localized with PDZD8-GFP associated with mCherry-Rab7 endosomes. Scale bar: 10 μm. c Endogenously expressed PDZD8 is recruited to Rab7-late endosomes. Immunofluorescence analysis of U2OS cells (upper panel) versus U2OS cells expressing mCherry-Rab7 (red). Cells were fixed, incubated with an anti-PDZD8 (green) and secondary anti-rabbit AlexaFluor488 antibodies and stained with Hoechst stain (blue, nucleus). Scale bar: 10 μm. d Rab7 is enriched in native PDZD8 and Protrudin immunoprecipitates. Mass-spectrometry analysis of Rab7 in immunopurifications of PDZD8 from crosslinked HCT116 cell extracts and human fibroblasts (left), and of Protrudin from crosslinked HCT116 cells and HEK293 cells (right). Rab7 intensity values are log2 label-free quantification (LFQ). One sided Student t test was performed between the control (no antibody) and indicated antibody with permutation-based FDR q value < 0.05 and S0 = 0.1. * and ** refer to FDR q value <0.05 and 0.01, respectively. Data presented as mean values ± S.D. Three biological replicates were performed. P-values: 0.0045 (FDR q-value 0.044; test statistic −3.477) and 0.0015 (FDR q-value 0.0497; test statistic −1.139) for PDZD8 vs control; HCT116 and fibroblasts, respectively. P-values: 0.0007 (FDR q-value 0.006; test statistic −5.797) and 0.0326 (FDR q-value 0.0486; test statistic −2.223) for Protrudin vs control; HCT116 and HEK293, respectively. Source data are provided as a Source Data file. e Rab7-GTP is required for recruitment of PDZD8-GFP to late endosomes. U2OS cells co-expressing PDZD8-GFP and mCherry-tagged Rab7 T22N (top) and Q67L (bottom) mutants. Scale bar: 10 μm. f Analysis of PDZD8-GFP and Rab7 interaction by immunoprecipitation. Extracts from HEK293T cells expressing PDZD8-GFP or PDZD8ΔCC-GFP, and wildtype mCherry-Rab7 or mCherry-Rab7 T22N or Q67L mutants were immunoprecipitated using anti-GFP-Trap beads and western analysis was performed using anti-GFP, anti-Actin and anti-Rab7 antibodies for detection of PDZD8-GFP, Actin, and mCherry-Rab7 respectively.

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