Role of myristoylation in membrane attachment and function of Gα_i-3 on Golgi membranes

Heterotrimeric G protein α-subunits localized on the cytoplasmic face of Golgi membranes are involved in regulating vesicle trafficking and protein secretion. We investigated the role of myristoylation in attachment of the Gα_i-3 subunit to Golgi membranes. Gαi-3 was epitopetagged by insertion of a FLAG sequence at an NH₂-terminal site predicted to interfere with myristoylation, and the result- ing NT-α_i-3 construct was stably transfected and expressed in polarized epithelial LLC-PK₁ cells. Metabolic labeling confirmed that the translation product of NT-α_i-3 was not myris- toylated. In contrast to endogenous Gα_i-3, which is tightly bound to Golgi membranes, the unmyristoylated FLAG- tagged NT-α_i-3 did not attach to membranes; it was localized by immunofluorescence in the cytoplasm of LLC-PK₁ cells and was detected only in the cytosol fraction of cell homogenates. Pertussis toxin-dependent ADP-ribosylation was used to test the ability of NT-α_i-3 to interact with membrane-bound γβ-subunits. In both in vitro and in vivo assays, cytosolic NT-α_i-3 alone was not ADP-ribosylated, although in the presence of membranes it could interact with Gβγ-subunits to form heterotrimers. The expression of NT-α_i-3 in LLC-PK₁ cells altered the rate of basolateral secretion of sulfated proteoglycans, consistent with the demonstrated function of endogenous Gα_i-3. These data are consistent with a model in which Gα_i-3 utilizes NH₂-terminal myristoylation to bind to Golgi membranes and to maximize its interaction with Gβγ- subunits. Furthermore, our results show that stable attachment of Gα_i-3 to Golgi membranes is not required for it to participate as a regulatory element in vesicle trafficking in the secretory pathway.