LAMTOR controls endosomal positioning by negatively regulating the BORC–Arl8b interaction. (A) The interaction of Arl8b-SH with the BORC subunit lyspersin is increased in LAMTOR1 KO cells. Strep-purified Arl8b-SH or HS-EGFP from LAMTOR1 KO and WT cells were analyzed by immunoblotting. (B) Results of four biologically independent experiments were quantified. Mean ± SD. **, P ≤ 0.01; P = 0.0059. (C) In lyspersin KO cells, the irreversible association of BORC with LAMTOR, achieved by Venus fluorophore reconstitution (lyspersin-VF1, LAMTOR1-VF2), immobilizes LAMP1-positive endosomes in the perinuclear region. No interaction (Venus signal) was detected in cells expressing lyspersin-VF1 and the control Zipper-VF2. Lyspersin-VF1 expression in lyspersin KO cells restores transportation of late endosomes toward the cell periphery. The overexpression of two LAMTOR subunits (LAMTOR3-VF1 and LAMTOR1-FV2) results in Venus-fluorophore reconstitution, indicating an interaction between both proteins in lyspersin KO and WT cells. The Venus signal in peripheral LAMP1 vesicles is found exclusively in WT cells. Analysis of Venus-fluorophore reconstitution (green) and indirect IF of endogenous LAMP1 (red). Representative images are shown. Arrows indicate endosomes positive for LAMP1 and BORC–LAMTOR irreversible interaction (Venus). Arrowheads show LAMP1-positive but BORC–LAMTOR interaction–negative late endosomes. Dashed line indicates cell borders. Representative images are shown. Bars: 10 µm; (inset) 2 µm. Legend: yellow trapezoid, LAMTOR complex; dark blue trapezoid, BORCΔlyspersin; light blue shape with discrimination of individual domains (N-domain, PRR, and C-domain), lyspersin; white ellipse, Zipper (negative control); white semicircles, VFs; two green semicircles together, Venus reconstitution and interaction between tagged proteins. The adjacent cell model depicts the presence (green) or absence of interaction (gray) and the overall position of late endosomes in the transfected cells.