THE G-PROTEIN GAMMA-SUBUNIT - REQUIREMENTS FOR DIMERIZATION WITH BETA-SUBUNITS

Guanine nucleotide-binding protein beta and gamma subunits form a tigh tly 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 l ysate and then assembled into dimers, but beta(1) cannot form beta gam ma 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 ai d of specific chaperonins or other cofactors. Dimerization-competent g amma(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 de fine the part of gamma(2) that is needed for native beta gamma dimer f ormation, a series of N- and C-terminal truncations were generated, sy nthesized in vitro, and incubated with beta(1). Dimerization was asses sed by stabilization of beta(1) to tryptic proteolysis. Truncation of up to 13 amino acids at the C terminus did not affect dimerization wit h B-1, whereas removal of 27 amino acids prevented it, Therefore, a re gion between residues 45 and 59 of gamma(2) is important for dimerizat ion, Truncation of 15 amino acids from the N terminus greatly diminish ed the formation of beta gamma dimers, while removal of 25 amino acids entirely blocked it, Thus, another region important for forming nativ e beta gamma is near the N terminus. Extension of the N terminus by 12 amino acids that include the influenza virus hemagglutinin epitope di d not prevent beta gamma dimerization, Furthermore, in intact S-35-lab eled COS cells, epitope-tagged gamma(2) coimmunoprecipitates with beta and alpha subunits. The N-terminal epitope tag must lie at the surfac e of the heterotrimer since it prevents neither heterotrimer formation nor access of the antibody.