Reconstitution of cyc− S49 membranes by in vitro translated Gsα Membrane anchorage and functional implications

AbstractAfter ADP-ribosylation by cholera toxin which promotes dissociation of the subunits, the α-subunit of Gs(Gsα) remained strongly associated with plasma membranes of wild-type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH ≥ 11.5. In vitro translated Gsα could interact with plasma membranes from the cyc− mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTPγS did not alter anchorage of Gsα. The characteristics of the association of in vitro translated Gsα with cyc− membranes after GTPγS treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP-ribosylated form in wild-type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc− membranes by in vitro translated α-subunit of Gs