CHARACTERIZATION OF MAMMALIAN G(S)-ALPHA PROTEINS EXPRESSED IN YEAST

The guanine nucleotide regulatory protein, G(S), mediates transmembran e signaling by coupling membrane receptors to the stimulation of adeny lyl cyclase activity. The full length coding sequences for the M(r) = 42-45,000, short form (S), and M(r) = 46-52,000, long form (L), of the alpha-subunits of rat G(S) were placed in yeast expression vectors un der the regulatory control of the copper-inducible CUP1 promoter and t ransformed into Saccharomyces cerevisiae. in the presence of 100 mu M CuSO4, the transformed yeast expressed G(S)-alpha mRNAs and proteins. In reconstitution experiments, rat G(S)-alpha(S and L), solubilized fr om yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol-, and guanine nucleotide-dependent sensitivity to adenylyl cyclase cata lytic units in S49 lymphoma cyc(-) cell membranes, which are devoid of endogenous G(S)-alpha G(S)-alpha(S) demonstrated twice the activity o f G(S)-alpha(L) in reconstitution assays of fluoride-stimulated adenyl yl cyclase activity. Comparison of G(S)-alpha(S) expressed in yeast wi th G(S) purified from rabbit liver or human erythrocytes showed that t he crude recombinant protein was fully competent in reconstituting NaF -stimulated adenylyl cyclase activity, but was only 2-5% as potent as purified G(S). Addition of bovine brain beta gamma subunits during rec onstitution enhanced all parameters of adenylyl cyclase activity for G (S)-alpha(S and L) obtained from yeast. In contrast, transducin beta g amma only enhanced agonist-stimulated adenylyl cyclase activity for G( S)-alpha(S and L) following reconstitution. These results demonstrate that the expression of functional mammalian G(S)-alpha subunits in yea st may be useful for their biochemical characterization.