Arl8b requires lyspersin but not LAMTOR for late endosomal/lysosomal localization. (A) Lyspersin is essential to recruit Arl8b to LAMP1-positive endosomes. Control WT, lyspersin KO, and rescue cell lines were transfected with Arl8b-EGFP. Representative, merged, and single-channel images of endogenous LAMP1 (red) and Arl8b-EGFP (green) are shown. Bars: 10 µm; inset, 2 µm. (B) Arl8b recruitment to late endosomes/lysosomes is LAMTOR-independent. Control WT, LAMTOR1 KO, and rescue cell lines were transfected with Arl8b-EGFP. Experiment was performed as in A. Representative images are shown. Bars: 10 µm; inset, 2 µm. (C) The C-domain of lyspersin or truncation mutants containing the C-domain are sufficient to restore the recruitment of Arl8b to LAMP1-positive endosomes in lyspersin KO cells. Lyspersin KO cells were cotransfected with Arl8b-EGFP and truncation mutants of lyspersin (Fig. 2 B). Experiment was performed as in A. Representative images are shown. Bars: 10 µm; (inset) 2 µm.