Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas

Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha(s), G alpha(i3) and G alpha(q/11) are present in Golgi fractions which are >95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha(s) was largely restricted to TGN-enriched fractions by immunoblotting, whereas G alpha(i3) and G alpha(q/11) were broadly distributed across Golgi fractions. G alpha(s) did not colocalize with TGN38 or caveolin, suggesting that G alpha(s) is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha(s) and G alpha(i3) were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha(s), G alpha(q/11), and G alpha(i3) associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G beta subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).