Palmitoylation is required for membrane association of activated but not inactive invertebrate visual Gqα

Abstract

The invertebrate visual G protein, iGqα plays a central role in invertebrate phototransduction by relaying signals from rhodopsin to phospholipase C leading to membrane depolarization. Previous studies have shown reversible association of iGqα with rhabdomeric membranes regulated by light. To address the mechanism of membrane association we cloned iGqα from a Loligo pealei photoreceptor cDNA library and expressed it in HEK293T cells. Mutations were introduced to eliminate putative sites for palmitoylation at cysteines in positions 3 and 4. Membrane and soluble fractions were prepared from cells where iGqα was either activated or maintained in the GDP-bound form, followed by identification of iGqα through immunoblot analysis. The wild-type iGqα was entirely membrane-bound and shown to be post-translationally modified by palmitoylation. The mutant iGqα (C3,4A) was not palmitoylated yet it was found to be membrane-associated in the inactive state, however, approximately half of the protein became soluble when activated. These results suggest that palmitoylation is not required for membrane association of iGqα in the inactive state but is important in maintaining the stable membrane association of activated iGqα–GTP. The mechanism by which iGqα moves away from the membrane into the cytosol in response to prolonged light-stimulation in the native squid eye appears, therefore, to involve both activation and depalmitoylation processes.