The yeast exchange assay, a new complementary method to screen for Db1-like protein specificity: identification of a novel RhoA exchange factor

The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) o f the Dbl family remain to be identified. Here we report a new method: the yeast exchange assay (YEA), a rapid qualitative test to perform a wide rang e screen for GEF specificity. In this assay based on the two-hybrid system, a wild type GTPase binds to its effector only after activation by a specif ic GEF, We validated the YEA by activating GTPases by previously reported G EFs, We further established that a novel GEF, GEF337, activates RhoA in the YEA. GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F-a ctin stress fiber assembly in fibroblasts, characteristic of RhoA activatio n, (C) 2000 Federation of European Biochemical Societies. Published by Else vier Science B.V. All rights reserved.