Extended Data Fig. 5: Characterization of SopF interaction with ARF1 and modification of the V-ATPase.
a, Analyses of the ability of ARF1 effectors to disrupt SopF–ARF1 complex formation. SopF ∆N62 in complex with His-tagged ARF1 Q71L was immobilized on Ni-NTA beads, and then incubated with indicated molar ratio of the ARF-binding domains of GGA1 or ARHGAP21. b, The V-ATPase complex used for in vitro modification by SopF. 293T cells stably expressing ATP6AP2-Flag were subjected to anti-Flag immunoprecipitation and analyzed by immunoblotting. c, d, Optimization and analyses of the reconstituted V-ATPase ADP-ribosylation by SopF. The assay was performed similarly as in Fig. 4e with omission of an indicated component (c) or in the presence of indicated molar ratio of the ARF-binding domains of GGA1, GGA3 or ARHGAP21 (d). Proteins in the reaction in d were shown by a Coomassie-stained SDS-PAGE gel. e, Mass spectrometry determination of molecular weight of His-ARF1 expressed alone or co-expressed with SopF in E. coli. Theoretical mass for the methionine-removed form of ARF1 is 24221.26 Da. f, 293T cells were transfected with ARF1-3×Flag alone or together with IpaJ or SopF. ARF1-3×Flag was immunopurified and its total molecular mass was determined by mass spectrometry. As a control, co-expression with IpaJ resulted in a 267.45-Da mass loss in ARF1 due to cleavage of the N-myristoylated glycine. Data are representative of two independent experiments.
