Bud3 has a GEF activity for Cdc42. (A) Two-hybrid assays between Cdc42 and Bud3. Two independent transformants of DNA-binding domain (DBD)–Bud3(m) and each activation domain (AD) fusion of WT or mutant Cdc42 or a vector control were patched on SGal-His and Trp (left) or SGal-His, Trp, and Leu (right) in the RSR1 or rsr1Δ host strain and incubated at 25°C for 4–5 d. (B) GST pull-down assays using MBP-Bud3(s) and extracts carrying each GST-Cdc42. Cdc42 and Bud3 were detected with anti-GST and anti-MBP antibodies, respectively. Numbers indicate relative amounts of Bud3 recovered, normalized to its recovery with GST-Cdc42^WT. (C) A representative GEF assay using GST-Cdc42 preloaded with mant-GDP. Mant-GDP fluorescence was measured over time after adding an equimolar amount of MBP-Bud3, MBP-Bud3ΔCR2, or MBP. (right) Mant-GDP fluorescence at t = 2,000 s is depicted from independent assays, normalized to the initial fluorescence (at t = 0). (D) A GEF assay using GST-Rsr1 preloaded with mant-GDP and MBP-Bud3 or MBP. Results from two independent assays were almost identical. au, arbitrary unit.