Fig 1: ACBD3 is a novel binding partner of KDELR1. A Volcano plot representing results of the label-free BioID of KDELR1. The logarithmic ratio of protein intensities in the KDELR1-BioID/KDELR1-myc were plotted against negative logarithmic p-values of the t test performed from the triplicates. Annotated Golgi proteins are candidate interaction partners of KDELR1 with a p-value of ratio significance <0.05 and marked with red dots. B Table summarizing annotated function of the Golgi proteins identified in KDELR1-BioID experiments. C Several Golgi-related proteins co-immunoprecipitate with KDELR1-mCherry. The protein extracts from HeLa cells transfected with mCherry tagged Sorting Nexin 3 (mCherry-SNX3 as a control) or KDELR1-mCherry were immunoprecipitated with anti-RFP agarose beads. These lysates and the immunoprecipitates (anti-RFP IPs) were analyzed by western blotting using anti-mCherry antibody and antibodies against the indicated Golgi proteins. D Split-ubiquitin-based membrane yeast two-hybrid assays suggest that KDELR1 interact with ACBD3 and ARFGAP1/3, but not with ARFGAP2 and Golgin97. Growth of yeast cells expressing KDELR1-Cub-LexA-VP16 bait or a pBT3-SUC bait vector with the following NubG-HA-preys: ACBD3, ARFGAP1, ARFGAP2, ARFGAP3, Golgin97, pPR3-N (prey vector as a negative control), and Ost1-NubI (positive control) were challenged on agar plates depleted of tryptophan and leucine (DDO plates, upper panel) or depleted of tryptophan, leucine, histidine, and adenine (QDO plates, bottom panel), plus 40 µg/ml X-a-Gal by spotting three independent transformants on different plates. The bait pTSU2-APP and the prey pNubG-Fe65 were used as positive controls in the MYTH assay. Experiments were repeated twice. D, E Interaction between ACBD3 and KDELR1 detected by split-YFP assays. HeLa cells were transfected with nYFP-3xFLAG-ACBD3 and KDELR1-myc-cYFP for 18 h before cells were fixed and observed by confocal microscopy (E). Single plasmid (nYFP-3xFLAG-ACBD3 or KDELR1-myc-cYFP) transfection was used as negative controls (D). White arrowheads indicate co-transfected cells, whereas white asterisks indicate cells transfected with a single plasmid, serving as a negative control. Scale bars = 10 µm