Fig. 3

ACBD3 is required for Golgi distribution of the three isoforms of KDELR at the Golgi. A Super-resolution 3D-SIM images showing a high degree of co-localization between endogenously tagged KDELR1-3xFLAG-mCherry and ACBD3, ARFGAP1, or ARFGAP3, and low degree of co-localization between the endogenously tagged KDELR1 and Golgin97 (negative control). Line profiles through regions of interest were analyzed by Fiji. Scale bars = 2 μm. Co-localization (Pearson’s R) was determined and subjected to two-tailed, unpaired t tests (n = 20 cells/combination, mean and SD, ****, p < 0.0001). B Confocal micrographs of HeLa cells co-expressing KDELR1-mCherry with deletion mutants of EGFP-ACBD3 after siRNA-mediated ACBD3 knockdown, showing that the GOLD domain of ACBD3 is required for retention of KDELR1-mCherry at the Golgi. For this experiment, we sequentially deleted the ACBP domain, the CC domain, the GOLD domain, or both ACBP+CC domains and used these constructs in the knockdown and rescue experiments. C Histogram summarizing the rescue experiments using full length EGFP-ACBD3, EGFP-ACBD3ΔACBP, EGFP-ACBD3ΔCC, EGFP-ACBD3ΔGOLD, EGFP-ACBD3-GOLD (ΔACBP+CC), respectively. Note that rescue with full length ACBD3 actually led to ~25% increase in the percentage of Golgi-localized KDEL-R1-mCherry over the WT control cells. In addition, we observed that EGFP-ACBD3 expression often abolished ER-localization of over-expressed KDELR1-mCherry entirely. (***p < 0.001; n.s., not significant) D EGFP-tagged GOLD domain of ACBD3 co-immunoprecipitates with KDELR1-mCherry. KDELR1-mCherry was co-transfected with EGFP-ACBD3 WT, EGFP-ACBD3ΔACBP, EGFP-ACBD3ΔCC, EGFP-ACBD3ΔGOLD, EGFP-GOLD domain, respectively, followed by cell lysis and immunoprecipitation using anti-RFP agarose beads. Experiments were repeated three times and representative western blots are shown here. Scale bars = 10 μm