Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection
(A) LegG1 promotes RanBP1 accumulation on LCVs. D. discoideum producing RanBP1-GFP (green) was infected (MOI 50, 1 h) with DsRed-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR077 (red), or with ΔlegG1/pER5 (M45-LegG1) and immuno-stained for SidC (blue). The percentage of RanBP1-GFP-positive LCVs (n = 100/strain, 5 independent experiments) was scored in lysates of infected cells (**, p<0.01; ***, p<0.001). (B) Production of Ran(GTP) in infected macrophages. RAW264.7 macrophages were infected (MOI 25, 1 h) with L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR033 (vector) or with ΔlegG1/pSU19 (M45-LegG1). The infected macrophages were lysed, activated Ran was immuno-precipitated with an antibody specifically recognizing Ran(GTP) and visualized by Western blot using an anti-Ran antibody. Lysates of uninfected cells incubated with GTP or GDP in presence of EDTA were used as positive or negative controls for endogenous GEF activity. Loading control: Western blot of Ran in samples before immuno-precipitation. (C) Production of Ran(GTP) in cell lysates. A549 epithelial cells were lysed and incubated with purified His6-LegG1 (native or heat-inactivated, h. i.) in presence of excess GTP (100 µM) or GDP (1 mM). Activated Ran was immuno-precipitated with an antibody specifically recognizing Ran(GTP) and visualized by Western blot using an anti-Ran antibody. Loading control: Western blot of Ran in samples before immuno-precipitation. The relative amount of Ran(GTP) was determined by densitometry; means and standard deviations of 4 (B, C) independent experiments are shown (*, p<0.05; **, p<0.01).