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The yeast exchange assay, a new complementary method to screen for Dbl‐like protein specificity: identification of a novel RhoA exchange factor
Author(s) -
De Toledo M.,
Colombo K.,
Nagase T.,
Ohara O.,
Fort P.,
Blangy A.
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01953-0
Subject(s) - rhoa , guanine nucleotide exchange factor , gtpase , effector , microbiology and biotechnology , rac1 , cdc42 , yeast , stress fiber , chemistry , biology , biochemistry , signal transduction , focal adhesion
The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of 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 range 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 specific GEF. We validated the YEA by activating GTPases by previously reported GEFs. We further established that a novel GEF, GEF337, activates RhoA in the YEA. GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F‐actin stress fiber assembly in fibroblasts, characteristic of RhoA activation.