Fig 1: RABGAP1 interacts with TUFT1 and regulates lysosomal positioning and mTORC1 signaling.a Lysates of A549 cells transfected with the indicated siRNAs were subjected to immunoprecipitation using HA or TUFT1 antibodies. Immunoprecipitates and 3% input extracts were immunoblotted with the indicated antibodies. Results are representative of two independent experiments. b GST pull-down assays were performed with GST-10xHis-TUFT1, negative control GST, and recombinant RABGAP1. Specific interaction of these proteins was detected by immunoblotting. The lower panel demonstrates the comparable GST fusion protein loading on the lanes. CBB, Coomassie Brilliant Blue staining. c A549 cells transfected with the indicated siRNAs were starved (3 h) or starved and restimulated (10 min) with nutrients. Cell lysates were analyzed by immunoblotting. The relative amount of phosphorylated protein was quantified as in Fig. 2b. Results are representative of two independent experiments. d A549 cells transfected with the indicated siRNAs were starved (3 h) and restimulated (10 min) with nutrients, then immunostained with antibodies for LAMP2 (red) and mTOR (green). The merged images are presented in the bottom row. Images are representative of two independent experiments. Scale bar, 10 μm. e TUFT1 modulates lysosomal positioning, vesicular trafficking and mTORC1 signaling through its interactions with RABGAP1. A working model illustrating the role of TUFT1-RABGAP1 in lysosomal positioning, vesicular trafficking and mTORC1 activation.