Figure 5.

The RabGAP activity of LepB from L. drancourtii. (A) In vitro GAP assay of LepB from L. drancourtii using Rab1 as the substrate. LepB^drancourtii is the purified GAP domain of LepB from L. drancourtii (residues 316-620). Arg447 and Glu452, corresponding to Arg444 and Glu449 in L. pneumophila LepB, were subjected to mutation analysis in this assay. (B) Effects of Arg447 and Glu452 mutations on AlF_n-mediated LepB^drancourtii-Rab1-GDP complex formation. The assay was performed similarly as that in Figure 1C. Shown at the top are the Superdex-75 gel filtration chromatograms colored accordingly. Peek 1, 2, and 3 correspond to LepB^drancourtii-Rab1-GDP-AlF_n complex, free LepB^drancourtii, and free Rab1, respectively. Elution fractions in the indicated range were analyzed by SDS-PAGE and Coomassie blue-stained gels are shown at the bottom.