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 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).
Denker
S P
SP
Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, 92093 USA.
McCaffery
J M
JM
Palade
G E
GE
Insel
P A
PA
Farquhar
M G
MG
D020727:ADP-Ribosylation Factors; D000818:Animals; D001693:Biological Transport, Active; D051242:Caveolin 1; D022461:Caveolins; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D010179:Pancreas / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors