Fig. 4

Photoactivation experiments reveals that knockout of ACBD3 results in acceleration of KDELR1 retrograde transport. A, B HeLa or HeLa-ACBD3-KO cells were co-transfected with KDELR1-FM4-SNAP and ST-RFP. After D/D solubilizer treatment, ER-Golgi antegrade transport of KDELR1-FM4-SNAP is monitored by live cell imaging acquired every 30 s for 10 min. Imaging sequences at the indicated time points are presented here. Scale bars = 10 μm. C To measure the amount of KDELR1 outflux from the Golgi, WT, or ACBD3-depleted HeLa cells were co-transfected with sialyltransferase-RFP (ST-RFP, a Golgi marker) and photoactivatable KDELR1-PA-GFP plasmids for 18 h. The KDELR1-PA-GFP in the Golgi were then activated by selecting an ROI of ST-RFP-positive region for intense 405-nm laser irradiation and the transport out of the Golgi are monitored by live cell imaging acquired every 5 s for 5 min. Imaging sequences prior to photoactivation (−10 s), immediately after photoactivation (0 s) and the indicated times following photoactivation are presented here. Magnified regions of interest (indicated by white boxes) from WT and ACBD3-KO cells at 100 s time point shows Golgi-derived tubules which are highlighted by white arrowheads. Scale bars = 10 μm. D The intensity of photoactivated GFP remaining in the Golgi area was expressed as a percentage of the intensity of photoactivated GFP at time point 0 and plotted as a function of time. The integrated fluorescence over the entire cell was normalized to the integrated fluorescence at time point 0 as a function of time (green and black lines), showing the photobleaching effect during live imaging, serving as a control for fluorescence (n = 22). (***p < 0.001) E Co-immunoprecipitation experiments indicate that ACBD3 depletion results in increased association of p24, ArfGAP1, coatomer, and Arf1 with KDELR1-mCherry.