Arl8b drives LAMP-positive membranes to mito-VPS41. (A) Confocal image of HeLa^VPS41KO cells cotransfected with mito-VPS41^WT-V5 and RILP-GFP. Cells were immunolabeled for a V5 tag. Scale bars: 20 µm. (B) Confocal microscopy of HeLa^VPS18KO and HeLa^VPS39KO cells cotransfected with Arl8b-HA and mito-VPS18-GFP or mito-VPS39-GFP as indicated in the figure. Immunostaining for an HA tag was performed. Scale bars: 20 µm. (C and D) Statistical quantification of Arl8b-HA recruitment on mitochondria harboring mito-VPS18-GFP or mito-VPS39-GFP as indicated. Statistical significance was obtained using an unpaired t test, ns: not significant. n > 25, mean ± SEM. (E) Confocal images of HeLa^VPS41KO cells single-transfected with mito-VPS41^WT-V5 or mito-V5. Cells were immunolabeled for a V5 tag and endogenous LAMP1. Scale bars: 20 µm. (F) Confocal images of HeLa^VPS41KO cells cotransfected with Arl8b-HA and mito-VPS41^WT-GFP or mito-GFP. Cells were immunolabeled for an HA tag and LAMP2A. Scale bars: 20 µm. (G) Statistical quantification of LAMP2A recruitment to mitochondria bearing mito-VPS41^WT-GFP or mito-GFP. Unpaired t test comparison was used to determine statistical significance, ****P < 0.0001. n > 25, mean ± SEM. (H) Quantification of LAMP1 redistribution to mito-VPS41^WT-V5 mitochondria in the presence (scrambled) or absence (siArl8b) of Arl8b. Data represent the mean ± SEM. Unpaired t test comparison was used to determine statistical significance, ****P < 0.0001. n > 25. (I) Immunoblot showing Arl8b expression in HeLa^VPS41KO treated with siArl8b or scrambled RNA. Actin was used as a loading control. (J) Fluorescence images of LAMP1 relocalization to mito-VPS41^WT-V5 upon depletion of endogenous Arl8b (top) or non-targeting RNA (scrambled, bottom). Scale bar: 20 µm. (K) Confocal images of LAMP1 relocalization to mito-VPS41^WT-GFP in HeLa^Arl8a-bKO cells rescued or not with Arl8b-HA. Scale bars: 20 µm. (L) Confocal image of HeLa^VPS41KO cells expressing mito-VPS41^∆WD40-GFP and VPS18-Flag and immunostained for a Flag tag. Scale bars: 20 µm. (M) Confocal images of HeLa^Arl8a-bKO cells stably expressing either VPS41^WT-GFP (top row) or H2B-NeonGreen (used as a negative control, bottom row). As a negative control, transiently overexpressed VPS41^WT-V5 was used. Cells were labeled for endogenous LAMP1 and V5. Scale bar: 20 and 5 µm (inset). (N) Immunoblot showing the comparison of VPS41 and Arl8b protein levels between HeLa^WT versus HeLa^Arl8a-bKO cells stably expressing either VPS41^WT-GFP or H2B-NeonGreen. Actin was used as a loading control. Source data are available for this figure: SourceData FS2.