ROLES OF LIPID MODIFICATIONS OF TRANSDUCIN SUBUNITS IN THEIR GDP-DEPENDENT ASSOCIATION AND MEMBRANE-BINDING

Transducin is an unusually soluble and dissociable heterotrimeric G-protein, although its T alpha and T beta gamma subunits are N-acylated and farnesylated, respectively. These lipid modifications have been suggested to contribute directly to the GDP-dependent T alpha-T beta gamma association, through specific lipid recognition sites on both protein subunits. We studied the dependence of subunit association on their bound lipids and on the presence of different lipidic environments. Association of native N-acylated (T-n alpha) or acyl-free recombinant (T-r alpha) T alpha with fainesylated and carboxymethylated (T-fc beta gamma), farnesylated (T-f beta gamma), or farnesyl-free (T-df beta gamma) TP beta gamma was analyzed by gradient centrifugation and gel filtration in the presence of detergent or phospholipid-cholate micelles and by cosedimentation with phospholipid vesicles. Without detergent, T-n alpha GDP and T-fc beta gamma associate only weakly in solution. The loss of T alpha acyl or T beta gamma famesyl residues induces total dissociation. With detergent or lipids; isolated T-fc beta gamma binds tightly to micelles or vesicles, while T-df beta gamma does not; T-n alpha GDP binds weakly, while deacylated T-r alpha GDP does not bind at all; and T-n alpha GDP binds cooperatively with T-fc beta gamma, while, T-r alpha GDP does not. Thus (i) the T alpha acyl chain binds weakly, whereas the T beta gamma farnesyl chain binds strongly to membrane lipids; (ii) there is no evidence for binding of the T alpha acyl chain to a polypeptide site in T beta gamma, nor for binding of the T beta gamma farnesyl chain to a polypeptidic site in T alpha, but the T alpha acyl chain seems to bind cooperatively with the T beta gamma farnesyl chain in the membrane lipids; (iii) the insertion of the two protein-attached lipids into the same membrane could contribute to the association of both subunits by favoring collision coupling of the properly oriented protein moieties on the membrane surface.