Interaction of RhoGDIs with Rho GTPases. A. Two-hybrid interactions between TbRhoGDI or LyGDI and the indicated Rho GTPases. After co-transformation of the corresponding plasmids into the Y190 yeast strain, colonies were allowed to grow on -Trp/-Leu plates and interactions verified and in colony-lift β-galactosidase assays. DdRac1b and DdRac1c behaved like DdRac1a (not shown). B. Translocation of TbCdc42 by TbRhoGDI. A membrane fraction of HeLa cells expressing GFP-tagged TbCdc42 was incubated in the absence (-) or presence (+) of 40 μM purified bacterially expressed His-tagged TbRhoGDI. The membranes were sedimented by centrifugation and aliquots of the supernatants were subjected to SDS-PAGE. The aliquot of membrane fraction (M) corresponds to the percentage of the analyzed supernatant (SN). TbCdc42 was detected with an antibody reactive against GFP. In these experiments His-tagged TbRhoGDI was always recovered in the supernatant. C. Translocation of D. discoideum Rho GTPases by bovine RhoGDI1. Membrane fractions of insect cells infected with baculoviruses encoding the indicated Dictyostelium GST-tagged Rho GTPases were incubated in the absence (-) or presence (+) of 40 μM purified bacterially expressed His-tagged RhoGDI1. The membranes were processed as in B. GTPases were detected with an antibody reactive against GST. In these experiments His-tagged RhoGDI1 was always recovered in the supernatant.