The yeast exchange assay, a new complementary method to screen for Dbl-like protein specificity: identification of a novel RhoA exchange factor.
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.
De Toledo
M
M
Centre de Recherches en Biochimie Macromoléculaire, CNRS UPR 1086, Montpellier, France.
Colombo
K
K
Nagase
T
T
Ohara
O
O
Fort
P
P
Blangy
A
A
D008841:Actin Cytoskeleton; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods*; D002384:Catalysis; D020662:Guanine Nucleotide Exchange Factors / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011518:Proto-Oncogene Proteins; D012441:Saccharomyces cerevisiae; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D020662:Guanine Nucleotide Exchange Factors; D011518:Proto-Oncogene Proteins; D020742:rhoA GTP-Binding Protein