Fig. 5

Mutational analysis of the N-terminal region of p18. a Ribbon models of α1 and α2 helices of p18 depicted on the molecular surface of RagC(RD) and MP1. Deletion boundaries tested in the cellular and in vitro experiments (NΔ50, NΔ55, and NΔ60) are indicated. b Schematic structures of N-terminal deletion mutants of p18. c p18 KO cells (KO) were transfected with the indicated mutants of p18, and subjected to immunostaining for p18, mTOR, 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. Complexes were eluted from 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 CBB staining