Purification, Reconstitution on Lipid Vesicles, and Assays of PDE6 and Its Activator G Protein, Transducin

PDE6 in rod and cone photoreceptors is the principal effector of phototransduction. It is kept at a very low activity level in the dark, and in the light it is strongly activated by the guanosine 5′-triphosphate-bound form of the α-subunit of the G protein, transducin. Both transducin and PDE6 are peripheral proteins, and understanding both their interactions with one another and the roles of lipids in their function requires reconstituting purified proteins on the surfaces of defined lipid bilayers. We describe here methods for purifying the proteins, reconstituting them with vesicles, and assaying catalytic activity and binding.