Fig. 4

Effects of p18 mutations on the assembly and function of Ragulator-RagA-C complex. a Ribbon model representation of the C-terminus of p18 depicted on the molecular surface of HBXIP, p14, p10, RagA(RD), and RagC(RD). Mutated amino acids and deleted regions tested in cellular and in vitro experiments (CΔ5, CΔ10, and CΔ15) are indicated. b Schematic structures of p18 mutants. Arrowheads indicate the locations of amino acid substitutions. c p18 KO cells (KO) were transfected with wild-type p18 (WT), C-terminal deletion mutants (CΔ5, CΔ10, and CΔ15), and mutants with multiple substitutions in the α2 helix (α2A) and the α4 helix (α4E), and subjected to immunostaining for mTOR, p18, and LAMP1. Arrowheads indicate the locations of lysosomal puncta. Dotted lines denote nuclei. d Total cell lysates prepared from the above cells were subjected to western blotting for the indicated proteins. e E. coli expressing p14, MP1, p10, HBXIP, and the indicated mutants of His-tagged p18 were lysed, and complexes were precipitated with HisTrap beads. Soluble lysates (s) and precipitates (p) were subjected to western blotting for the indicated proteins. f Ragulator was purified from the above precipitates and mixed with the purified RagA-C complex, and then reprecipitated with HisTrap beads. Soluble lysates (s) and precipitates (p) were subjected to SDS-PAGE, followed by Coomassie Brilliant Blue (CBB) staining