The DUF2365 domain mediates lyspersin recruitment to lysosomes via CE1 and Arl8b recruitment to lysosomes and lysosome dispersal via CE2. (A–G) The full-length or mutant GFP-lyspersin constructs indicated in the figure (see Fig. 2 B for scheme) were transiently expressed together with Arl8b-mCherry by transfection into lyspersin-KO HeLa cells. Fixed cells were immunostained with antibodies to GFP, mCherry, and LAMTOR4. WT (A) or lyspersin-KO (B) HeLa cells expressing Arl8b-mCherry were analyzed similarly as controls. Bar, 10 µm. Arrows point to lysosomes accumulated at peripheral sites. Pearson’s coefficients for the colocalization of GFP-lyspersin variants with Arl8b-mCherry are indicated in the merge images. Graphs on the right represent the distribution of lysosomes relative to the MTOC, normalized to the longest distance between the MTOC and the cell periphery. Values are the mean ± SD from 20 cells per condition. Note that overexpression of Arl8b-mCherry causes lysosomes to be more dispersed than in untransfected cells (Rosa-Ferreira and Munro, 2011; Pu et al., 2015).