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

• Published in: The Journal of cell biology Vol. 133; no. 5; pp. 1027 - 1040
• Main Authors: Denker, S P, McCaffery, J M, Palade, G E, Insel, P A, Farquhar, M G
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.06.1996; The Rockefeller University Press
• Subjects: ADP-Ribosylation Factors; Animals; Biological Transport, Active; Caveolin 1; Caveolins; Cell Membrane - metabolism; Cells; Cellular biology; Golgi Apparatus - metabolism; Golgi Apparatus - ultrastructure; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; Immunohistochemistry; Intracellular Membranes - metabolism; Male; Membrane Proteins - metabolism; Membranes; Microscopy, Immunoelectron; Pancreas; Pancreas - metabolism; Pancreas - ultrastructure; Protein Binding; Protein Conformation; Proteins; Rats; Rats, Sprague-Dawley; Rodents; Signal Transduction
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.133.5.1027

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 alpha 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).