Mende, U; Schmidt, CJ; Yi, F; Spring, DJ; Neer, EJ; ,
Guanine nucleotide-binding protein beta and gamma subunits form a tightly bound complex that can only be separated by denaturation, Assembly of beta and gamma subunits is a complicated process, The beta(1) and gamma(2) subunits can be synthesized in vitro in rabbit reticulocyte lysate and then assembled into dimers, but beta(1) cannot form beta gamma dimers when synthesized in a wheat germ extract, In contrast, gamma(2) translated in either system can dimerize with beta(1), suggesting that dimerization-competent gamma(2) can be synthesized without the aid of specific chaperonins or other cofactors. Dimerization-competent gamma(2) in solution forms an asymmetric particle with a Stokes radius of about 21 +/- 0.4 Angstrom (n = 4), s(20,w) of 0.9 S (range 0.8-1.0 S, n = 2), and frictional ratio of 1.57 (assuming no hydration), To define the part of gamma(2) that is needed for native beta gamma dimer formation, a series of N- and C-terminal truncations were generated, synthesized in vitro, and incubated with beta(1). Dimerization was assessed by stabilization of beta(1) to tryptic proteolysis. Truncation of up to 13 amino acids at the C terminus did not affect dimerization with B-1, whereas removal of 27 amino acids prevented it, Therefore, a region between residues 45 and 59 of gamma(2) is important for dimerization, Truncation of 15 amino acids from the N terminus greatly diminished the formation of beta gamma dimers, while removal of 25 amino acids entirely blocked it, Thus, another region important for forming native beta gamma is near the N terminus. Extension of the N terminus by 12 amino acids that include the influenza virus hemagglutinin epitope did not prevent beta gamma dimerization, Furthermore, in intact S-35-labeled COS cells, epitope-tagged gamma(2) coimmunoprecipitates with beta and alpha subunits. The N-terminal epitope tag must lie at the surface of the heterotrimer since it prevents neither heterotrimer formation nor access of the antibody.