Mito-VPS41 recruits biosynthetic LAMP2A-RUSH but not CD-M6PR-RUSH and CatD. (A) Confocal images of HeLa^VPS41KO cells stably expressing mito-mCherry-FRB transfected with FKBP-VPS41^WT-Halo and LAMP2A-RUSH after 45 min of treatment with biotin and rapalog (top) or only biotin (bottom) used as a negative control. Arrows indicate LAMP2-RUSH vesicles and their overlap with mito-mCherry-FRB images. Scale bar: 10 and 5 µm (inset magnification). (B) Scatter dot plot showing the quantification of the mitochondrion recruitment of LAMP2A-RUSH 45 min after biotin addition as shown in A. Quantification is expressed in fold change with value 1 given to LAMP2A-RUSH signal on the mask of the mitochondria expressing mito-mCherry-FRB not treated with rapalog used as a negative control. Statistical significance was calculated using an unpaired t test, **P < 0.01. n > 70, mean ± SEM. Grey dots and green dots represent individual cell data points and experimental replicate means, respectively. (C) Confocal images of HeLa^VPS41KO cells stably expressing mito-mCherry-FRB transfected with FKBP-VPS41^WT-Halo and LAMP2A-RUSH (top) or CD-M6PR-RUSH (bottom) 45 min after treatment with biotin and rapalog. Arrows indicate LAMP2-RUSH or CD-M6PR-RUSH vesicles and their overlap with mito-mCherry-FRB images. Scale bar: 10 and 5 µm (inset). (D) Quantification of mitochondrion recruitment of LAMP2A-RUSH and CD-M6PR-RUSH 45 min after biotin treatment as shown in C. Quantification is expressed in fold change with value 1 to CD-M6PR-RUSH signal on the mask of the mitochondria expressing mito-mCherry-FRB treated with rapalog and biotin used as a negative control. Statistical significance was calculated using an unpaired t test, *P < 0.01. n > 70, mean ± SEM. (E) Confocal images showing the CatD distribution in HeLa^VPS41KO expressing Arl8b-GFP together with either mito-VPS41^WT-V5 or negative control mito-V5. Scale bar: 20 µm.