Fig. 1: LOPIT-DC on mitochondrial relocated golgin-97 and GCC88.

a Applying proximity biotinylation to mitochondrially relocated golgin-97 identifies its interaction with TBC1D23 and the WDR11 complex on endosome-to-Golgi vesicles. b Overview of the LOPIT-DC work flow. Cells are gently lysed and fractionated by differential ultracentrifugation. The peptides of each fraction are then labelled with 11-plex TMT, pre-fractionated through high-pH reversed phase UPLC and then analysed by MS² or SPS-MS³. This gives the profiles of the proteome, which are then analysed as in d. Image is adapted from the LOPIT-DC methods paper²³, under a Creative Commons Attribution 4.0 International License [http://creativecommons.org/licenses/by/4.0/]. c Immunoblot of proteins from Flp-In 293 cells stably expressing the doxycycline-inducible mito, golgin-97-mito and GCC88-mito with 1 μg ml⁻¹ doxycycline for 48 h, representative of two independent experimental replicates. d Principle component analysis (PCA) projections for the LOPIT-DC showing organelle markers before classification, after SVM classification and after TAGM classification. e, f Results of MitoRatio versus Bayes Factor analysis comparing mito control to golgin-97-mito and GCC88-mito. Thresholds are based on experimentally confirmed hits (see text) (log₂(MitoRatio) ≥ 0.40, log_e(Bayes Factor) ≥ 14.0). Mito versus golgin-97-mito: 140 proteins in addition to golgin-97 were within threshold out of 5914 analysed proteins (3591 after pre-filtering), all 140 listed in Supplementary Data 2. Mito versus GCC88-mito: 16 proteins in addition to GCC88 were within threshold out of 5532 analysed proteins (3585 proteins after pre-filtering), all 16 listed in Supplementary Data 2. g Quantification of confocal micrographs of Flp-In 293 cells as used in LOPIT-DC (Supplementary Fig. 2a) measuring the ratio of the mean intensity of TGN46 at the mitochondria over its intensity at the Golgi (MitoRatio). Boxplots are of MitoRatios of all cells quantified across 3 independent experiments (n ≥ 114), and show the median, the first and third quartiles (Q1 and Q3), minimum (Q1-1.5 × interquartile range (IQR)), and maximum (Q3 + 1.5 × IQR). p-values are of two-sample two-sided t-tests comparing the overall mean MitoRatio of each replicate. Data are provided as a Source Data file. h, i Confocal micrographs of HeLa cells expressing the indicated golgin-mito construct (HA) stained for endogenous ATG9A or exogenous Furin-GFP, all micrographs representative of three independent experimental replicates. Scale bars, 10 μm.