Fig. 5

The cdc42 mutants defect in binding to Sec3N and Gic2N in vitro. a Amino acid sequence alignment of the S. cerevisiae Cdc42 and Rho1. Identical residues are indicated as asterisks. Switch I and II of Cdc42 and Rho1 are colored gray. Carrots point to amino acids in Cdc42 that have been mutated: positions 36, 37, 61, 64 and 66. b Schematic drawing of interactions between Sec3 and Rho1 (adapted from [32]), aligned beside the corresponding amino acid in Cdc42. c The cdc42 mutants were not able to bind to Sec3N in vitro. GST fusion proteins containing (a.a.71–241) of Sec3 was purified and conjugated to glutathione Sepharose. Cdc42, cdc42-301, cdc42-302, cdc42-303, cdc42-304 and cdc42-305 were expressed as a Hisx6 fusion and purified from bacteria. The in vitro binding assay was performed using GST-Sec3 and Hisx6-Cdc42 in the presence of GTPγS. The Hisx6-Cdc42 fusion protein bound to the GST-Sec3N Sepharose was detected by Western blotting with anti-Hisx6 antibody (bottom). Equal amounts of Sec3 fusion proteins on beads were used in the binding assay (middle; Ponceau S staining). Equal amounts of cdc42 wild type and mutants were used (top, 1% input, Western blotting with anti-Hisx6 antibody). The representative results from three independent experiments are shown. d The cdc42 mutants defect in binding to Gic2N in vitro. GST fusion proteins containing (a.a.1–155) of Gic2 were purified and conjugated to glutathione Sepharose. Cdc42, cdc42-301, cdc42-302, cdc42-303, cdc42-304 and cdc42-305 were expressed as a Hisx6 fusion and purified from bacteria. The in vitro binding assay was performed using GST-Gic2 and Hisx6-Cdc42 in the presence of GTPγS. The Hisx6-Cdc42 fusion protein bound to the GST-Gic2N Sepharose and was detected by Western blotting with anti-Hisx6 antibody (bottom). Equal amounts of Gic2 fusion proteins on beads were used in the binding assay (middle; Ponceau S staining). Equal amounts of Cdc42 wild type and mutants were used (top, 2% input, Western blotting with anti-Hisx6 antibody). The representative results from three independent experiments are shown