LAMP2A-RUSH biosynthetic vesicles are recruited by mito-VPS41. (A) Fluorescence image of HeLa^VPS41KO cells expressing mito-V5 and LAMP2A-RUSH after 1.5 h of incubation with biotin, used as a negative control for RUSH experiment in Fig. 10, A and B. Scale bar: 10 and 5 µm (inset). (B) Fluorescence image of HeLa^VPS41KO cells stably expressing mito-mCherry-FRB transfected with FKBP-VPS41^WT-Halo and LAMP2A-RUSH. Image shows localization of mito-mCherry-FRB (magenta) and FRB-VPS41^WT-FKBP (green) after 6 min of A/C heterodimerizer and biotin treatment (top). Representative time-lapse images of VPS41 recruitment to mitochondria (on the right). Scale bar: 10 and 5 µm (inset). (C) Fluorescence image at minute 6 of negative control where cells were only treated with biotin (top). Representative time-lapse images of VPS41 and mitochondria (on the right). Scale bar: 10 and 5 µm (inset). (D) Fluorescence image of negative control as above after 24 min of treatment with biotin (top). Representative time-lapse images of LAMP2A-RUSH–positive vesicles and mitochondria expressing mito-mCherry-FRB. No accumulation of LAMP2A on mitochondria was observed (bottom). Scale bar: 10 and 5 µm (inset). (E–G) Time-lapse panel of (E) FKBP-VPS41^WT-Halo and (G) LAMP2A-RUSH recruitment to mito-mCherry-FRB upon treatment with rapalog and biotin. (F–H) Time-lapse panel of (F) FKBP-VPS41^WT-Halo and (H) LAMP2A-RUSH and mito-mCherry-FRB upon treatment with only biotin. Scale bar: 10 µm.