Fig. 1: Identification of LD-mitochondria interface proteins using proximity proteomic screens.
a BioID targeted to the surface of LDs, containing the transmembrane domain of AAM-B, RFP (mScarlet), HA, and BirA* (AAMTMD-RFP-HA-BirA*). b BioID targeted to the surface of the outer membrane of mitochondria, containing the transmembrane domain of FIS1, CFP (mTurquoise2), FLAG, and BirA* (FIS1TMD-CFP-FLAG-BirA*). c Mass spectrometric analysis of biotinylated proteins purified from HepG2 cells stably expressing AAMTMD-RFP-HA-BirA*chimeras (LD-BioID). Plot compares BirA* to the negative control in HepG2 cells supplemented with biotin. Colored dots indicate proteins enriched using the three independent constructs (a–e). Proteins known to be enriched at LDs (ArtGAP1, SNX, FASN), peroxisomes (GOSAR1, ABCD3), endoplasmic reticulum (ESYT1, ESYT2, RTN4) and mitochondria (DNM1L, CSDE1, OCIAD1) are highlighted. d Mass spectrometric analysis of biotinylated proteins purified from HepG2 cells stably expressing Fis1TDM-CFP-FLAG-BirA* (Mito-BioID). Experiment and data analysis as indicated in (c). e Design of the Split-BioID approach, containing the transmembrane domain of FIS1, CFP, FLAG, and half of BirA*(BirAN*) targeted to the surface of the outer membrane of mitochondria (Fis1TMD-CFP-FLAG-BirAN*). The transmembrane domain of AAM-B anchors RFP, HA, and the other half of BirA*(BirAC*) targeted to the surface of LDs (AAMTMD-RFP-HA-BirA*). f Airyscan imaging of HepG2 cells stably expressing the Split-BioID. Merged image showing Fis1TMD-CFP-FLAG-BirAN* (magenta) and AAMTMD-RFP-HA-BirAC*(green). (representative of 4 independent experiments, scale bar, 2 μm). g Mass spectrometric analysis of biotinylated proteins purified from HepG2 cells stably expressing Split-BioID and negative controls (non-BirA*-transfected HepG2 cells). Colored dots indicate commonly enriched proteins in panels C & D. h Venn diagram showing the number of proteins enriched using Split-BioID, LD-BioID and mito-BioID approaches. i Dot-plot analysis of commonly enriched proteins using Split-BioID, LD-BioID and mito-BioID approaches. j Validation of proteins identified by BioID using an organelle coprecipitation assay of HepG2 cell lysate. LD-mitochondria fraction (LD+Mito), mitochondrial fraction (Mito), and cytosol fraction (Cyto) were examined by immunoblot. Representative of n = 3 biological replicates. For c, d, g and i, data analyzed with unpaired two-tailed Student’s t-test with FDR < 0.05, n = 4 biological replicates. See also Supplementary Fig. 1 and Supplementary Data 1. Created in BioRender. Keenan, S. (2025) https://BioRender.com/n75s805.
