BORC interacts with Ragulator on lysosomes. (A) Proteins that interact with BLOS2 (subunit of both BORC and BLOC-1), lyspersin (subunit of BORC), and pallidin (subunit of BLOC-1) tagged with One-STrEP-FLAG (OSF) or FLAG-One-STrEP (FOS) were identified by TAP-MS. Two independent analyses were performed for OSF-BLOS2 and OSF-lyspersin. Total peptide numbers of the coisolated proteins are shown. (B) OSF- or FOS-tagged BLOS2, lyspersin, pallidin, or LAMTOR1 were expressed by stable transfection in WT HeLa cells, and FOS-tagged myrlysin was expressed by stable transfection in myrlysin-KO HeLa cells. Cells were extracted with detergent-containing buffer and subjected to pull-down with Strep-Tactin beads followed by SDS-PAGE. Endogenous myrlysin and LAMTOR4, and the FLAG epitope, were detected by immunoblotting (IB). The positions of molecular mass markers (in kilodaltons) are indicated on the left. (C) Y2H analysis was performed by cotransforming yeast with plasmids encoding BORC subunits fused to the Gal4 binding domain (BD; top) and Ragulator subunits fused to the Gal4 activation domain (AD; left). In this and subsequent Y2H experiments, yeast transformants were grown on −His (top) or +His (bottom) plates. SV40 T antigen and p53 were used as controls. (D) GFP-lyspersin was transiently expressed by transfection in WT HeLa cells, and lysosomes were visualized by immunostaining with antibodies to endogenous LAMP1 and LAMTOR4. Bar, 5 µm. Magnifications of the boxed area are shown in the bottom row. Bar, 11 µm.