Fig. 5

ACBD3 depletion alters KDELR association with Arf1, promoting Arf1-dependent tubular carrier formation at the Golgi. A–C myc-ACBD3, ARFGAP1-myc, or ARFGAP3-myc can efficiently pull down KDELR1-mCherry, but myc-ACBD3 does not co-immunoprecipitate with the key components of the retrograde trafficking machinery. ARFGAP1-myc co-immunoprecipitates with γCOP, ARFGAP3, ARF1, and p24 proteins, while ARFGAP3-myc only co-immunoprecipitates with γCOP. HeLa cells were co-transfected KDELR1-mCherry with myc-ACBD3, ARFGAP1-myc, or ARFGAP3-myc, respectively, and the extracts were immunoprecipitated with a myc-trap agarose beads. The lysates and the immunoprecipitates were analyzed by western blotting, as indicated. D, E Kinetics of Arf1-EGFP binding to and dissociation from Golgi membranes in WT and ACBD3-KO HeLa cells. D Representative FRAP experiments in WT or ACBD3-KO HeLa cells transiently expressing Arf1-EGFP. The Golgi was selectively photobleached (white circle). The first frames show an initial prebleached image. After photobleaching, image frames were selected at times 10, 50, 100, and 300 s. Scale bar, 10 μm. E Quantification of Golgi intensity in Arf1-EGFP–expressing WT or ACBD3-KO HeLa cells. Statistical analysis was performed using two-tailed, paired t test (mean±SEM, N = 10, ****p < 0.0001). F HeLa WT, ACBD3-KD (stably knockdown) or ACBD3-KO cells were analyzed for levels of Arf1-GTP, as described in the methods. G Live-cell imaging of transiently expressed Arf1-EGFP in HeLa WT or ACBD3-KO cells. Zoomed Golgi areas were shown in the bottom panels. Examples of Golgi-derived ARF1-rich tubules are highlighted by arrowheads. H Confocal images of KDELR1^endo-3xFlag-mCherry in HeLa cells showing that depletion of PI4KB does not result in re-distribution of KDELR1^endo-3xFlag-mCherry to the ER in vivo. Scale bars = 10 μm. I Western blots showing depletion of PI4KB, by RNA interference. J Confocal images of KDELR1^endo-3xFlag-mCherry in HeLa cells showing that overexpression of PI4KB does not result in its re-distribution to the ER in vivo. K Golgi localization of KDELR1^endo-3xFlag-mCherry is unaffected by chemical inhibition of PI4K using PIK-93. Scale bars = 10 μm