Open AccessARD1, a 64-kDa bifunctional protein containing an 18-kDa GTP-binding ADP-ribosylation factor domain and a 46-kDa GTPase-activating domain.
Author(s) -
Nicolas Vitale,
Joel Moss,
Martha Vaughan
Publication year - 1996
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.93.5.1941
Subject(s) - heterotrimeric g protein , gtpase , gtpase activating protein , adp ribosylation factor , gtp' , g protein , biology , g alpha subunit , gtp binding protein regulators , biochemistry , guanosine triphosphate , binding domain , protein subunit , binding protein , binding site , receptor , enzyme , gene , cell , golgi apparatus
The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins (G proteins) hydrolyze GTP at a rate significantly higher than do most members of the Ras family of approximatelly 20-kDa GTP-binding proteins, which depend on a GTPase-activating protein (GAP) for acceleration of GTP hydrolysis. It has been demonstrated that an inserted domain in the G-protein alpha subunit, not present in the much smaller Ras-like proteins, is responsible for this difference [Markby, D. W., Onrust, R. & Bourne, H. R. (1993) Science 262, 1895-1900]. We report here that ARD1, a 64-kDa protein with an 18-kDa carboxyl-terminal ADP-ribosylation factor (ARF) domain, exhibited significant GTPase activity, whereas the ARF domain, expressed as a recombinant protein in Escherichia coli, did not. Addition of the 46-kDa amino-terminal extension (similarly synthesized in E. coli) to the GTP-binding ARF-domain of ARD1 enhanced GTPase activity and inhibited GDP dissociation. The kinetic properties of mixtures of the ARF and non-ARF domains were similar to those of an intact recombinant ARD1. Physical association of the two proteins was demonstrated directly by gel filtration and by using the immobilized non-ARF domain. Thus, like the alpha subunits of heterotrimeric G proteins, ARD1 appears to consist of two domains that interact to regulate the biological activity of the protein.