Simple purification and functional reconstitution of octopus photoreceptor G(q) which couples rhodopsin to phospholipase C

In invertebrate photoreceptors, illuminated rhodopsin activates multiple G proteins, which are assumed to initiate multiple phototransduction cascades. In this paper, we focused on one of the phototransduction cascades, which utilizes rhodopsin, a G(q)-like G protein, and phospholipase C (PLC). A G(q)-like G protein from octopus photoreceptors was successfully purified to apparent homogeneity as an active form by simple two-step chromatography. The purified G protein had an alpha beta gamma-trimeric structure consisting of 44-kDa alpha, 37-kDa beta, and 9-kDa gamma subunits. The 44-kDa alpha subunit was assigned to the G(q) class by western blot with antiserum against mammalian G(q) alpha and by partial amino acid sequencing of its proteolytic fragments. Light-dependent binding of GTP gamma S was observed when the purified octopus G(q) was reconstituted with octopus rhodopsin that had been integrated into phospholipid vesicles. Octopus G(q) activated PLC beta(1) purified from bovine brain dose-dependently in the presence,of AlF4-. Finally, light-and GTP-dependent activation of PLC beta 1 was observed in a reconstitution system consisting of octopus rhodopsin, G(q), and bovine PLC beta 1.