Fig. 3
Validation of the interactions among different Ragulator components. a Co-IP assays to validate the interactions of p18 with the other four components. FLAG-tagged CASTOR1, the cytoplasmic arginine sensor that has no direct interaction with the Ragulator or the Rag GTPases, was used as a negative control. The p1814–161 variant that lacks the N-terminal lipid-modification region was denoted as the wild-type (WT) protein. WT p18 was fused with a FLAG tag and the p18 variants were fused with a FLAG-GST tag to improve expression. b Co-IP assays to validate the interactions of p18 with C7orf59 and HBXIP. Mutation L119R or V132D on p18 significantly impairs the interactions with the other four components; as negative controls, mutations V118R and R134D exhibit no notable effect on the interactions because the two residues have no direct interactions with C7orf59-HBXIP. c Cellular co-localization of WT and mutant p18 with the other four components. The interacting regions of p18 for MP1, HBXIP, and C7orf59 are essential for the assembly of the Ragulator complex, but the region for p14 is not. Scale bar represents 10 μm
