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Human RhoA/RhoGDI complex expressed in yeast: Gtp exchange is sufficient for translocation of RhoA to liposomes
Author(s) -
Read Paul W.,
Liu Xiaopu,
Longenecker Kenton,
Dipierro Charles G.,
Walker Lori A.,
Somlyo Avril V.,
Somlyo Andrew P.,
Nakamoto Robert K.
Publication year - 2000
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.9.2.376
Subject(s) - rhoa , gtp' , gtpase , microbiology and biotechnology , biology , cdc42 , cytosol , small gtpase , adp ribosylation factor , g protein , guanine nucleotide exchange factor , gtp binding protein regulators , biochemistry , signal transduction , chemistry , endoplasmic reticulum , golgi apparatus , enzyme
The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post‐translationally processed and, when co‐expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP‐ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [ 3 H]GDP or partially with GTP in the presence of submicromolar Mg 2+ . The GTP‐RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 × 10 −5 s −1 , considerably slower than free RhoA. Hydrolysis followed pseudo‐first‐order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V‐RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP‐bound G14V‐RhoA/RhoGDI complex (but not the GDP form) into serum‐starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP‐bound‐RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP‐RhoA did not. These results show that GTP‐triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or membrane receptors.