Fig. 2: Proteomics screening identified a PDZD8–FKBP8 protein complex.
From: Mitochondrial complexity is regulated at ER-mitochondria contact sites via PDZD8-FKBP8 tethering
a Scheme of the immunoprecipitation and LC–MS/MS analysis using the Pdzd8-3× HA KI mice neocortices. The immunoprecipitates from the neocortices of Pdzd8-3× HA mice or the control littermates using an anti-HA antibody were subjected to the LC–MS/MS analysis. b Diagram describing the genomic sequence of Pdzd8-3× HA KI mice. The sequence of a 3× HA tag was knocked-in at the C-terminus of the Pdzd8 coding sequence. c Volcano plot of proteins differentially binding to the PDZD8-3× HA. FKBP8 is labeled in red. Protrudin and VAPA, which have been previously reported to interact with PDZD8, are labeled in blue. The plot represents data from three biological replicates. The p-value was calculated using an unadjusted two-tailed Student’s t-test. d Scheme of labeling proteins in the vicinity of endogenous PDZD8. A Biotin ligase TurboID fused to PDZD8 generates biotin–5′-AMP from biotin and ATP. The biotin–5′-AMP can covalently bind to proteins located within about 20 nm of endogenously expressed PDZD8-TurboID. e Diagram describing the genomic sequence of the PDZD8-TurboID KI HeLa cell. The sequence of TurboID-P2A-Neor was knocked-in at the C-terminus of the PDZD8 coding sequence. f Volcano plot of proteins differentially biotinylated with biotin in the PDZD8-TurboID KI HeLa cell. FKBP8 is labeled in red. Protrudin and VAPA, which have been previously reported to interact with PDZD8, are labeled in blue. The volcano plot represents three biological replicates. The p-value was calculated using an unadjusted two-tailed Student’s t-test. g Numbers of proteins highly enriched in the IP–MS (c) and TurboID-MS (f) are shown in a Venn diagram. Twelve proteins are commonly found in the two proteomes. Note that FKBP8 is the only protein annotated with mitochondrial localization. h, i Analysis of the interaction between endogenous FKBP8 and endogenous PDZD8-3× HA from the mouse neocortex (h), or endogenous PDZD8-Venus from NIH3T3 cells. Extracts from neocortex in Pdzd8-3×HA KI mouse (h) or Pdzd8-Venus KI NIH3T3 cells (i) were subjected to immunoprecipitation (IP) with antibodies to HA or GFP respectively. The resulting precipitates as well as the original tissue extracts (Total) were subjected to immunoblot analysis with antibodies to FKBP8, VAPA, MFN2, HA (h), GFP (i), and β-actin (i). Data are representative of three independent experiments. Source data are provided as a Source Data file.
