Role of myristoylation in membrane attachment and function of G alpha(1-3) on Golgi membranes

Heterotrimeric G protein a-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 G alpha(i-3) subunit to Golgi membranes. G alpha(i-3) was epitope-tagged by insertion of a FLAG sequence at an NH2-terminal site predicted to interfere with myristoylation, and the resulting NT-alpha(i-3) construct was stably transfected and expressed in polarized epithelial LLC-PK1 cells. Metabolic labeling confirmed that the translation product of NT-alpha(i-3) was not myristoylated. In contrast to endogenous G alpha(i-3), which is tightly bound to Golgi membranes, the unmyristoylated FLAG-tagged NT-alpha(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-alpha(i-3) to interact with membrane-bound beta gamma-subunits. In both in vitro and in vivo assays, cytosolic NT-alpha(i-3) alone was not ADP-ribosylated, although in the presence of membranes it could interact with G beta gamma-subunits to form heterotrimers. The expression of NT-alpha(i-3) in LLC-PK1 cells altered the rate of basolateral secretion of sulfated proteoglycans, consistent with the demonstrated function of endogenous G alpha(i-3). These data are consistent with a model in which G alpha(i-3) utilizes NH2-terminal myristoylation to bind to Golgi membranes and to maximize its interaction with G beta gamma-subunits. Furthermore, our results show that stable attachment of G alpha(i-3) to Golgi membranes is not required for it to participate as a regulatory element in vesicle trafficking in the secretory pathway.