The LAMTOR–BORC interaction is lyspersin-dependent. (A) Deletion of lyspersin abolishes the interaction between LAMTOR and the remaining BORC subunits (BORCΔlyspersin). In contrast, the LAMTOR-RagA interaction remains unaffected. HA immunoprecipitates from control, WT, and lyspersin KO HeLa cells stably expressing LAMTOR1-HA were analyzed by immunoblotting. (B) Annotated representation of lyspersin truncation mutants. N-domain, N-terminal region; C-domain, C-terminal region. (C) Lyspersin interacts with the LAMTOR complex through its PRR whereas the C-domain of lyspersin is responsible for the interaction with the remaining BORC subunits. The DUF2365 domain is sufficient to mediate the interaction with both LAMTOR and BORCΔlyspersin. Immunoblotting analysis of Strep coprecipitates from lyspersin truncation mutants inducibly expressed in HEK293 Flp-In T-REx cells.