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

Abstract

Abstract. 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 ffactionation and immunocytochemical localization to investigate the distribution of Get and Gi3~/subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that Gas, Goti3 and Gotq/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 Got subunits. Gots was largely restricted to TGN-enriched fractions by immunoblotting, whereas Goti3 and Gotq/n were broadly distributed across Golgi fractions. Gets did not colocalize with TGN38 or caveolin, suggesting that Gets is associated with a distinct population of membranes. G[3 subunits were barely detectable in purified Golgi fractions.

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

“…The observation that paraformaldehyde fixation partially permeabilizes cells has been noted by others and is supported by immunofluorescence labeling of the Golgi apparatus matrix protein, GM-130. GM-130 is not labeled in intact (unfixed) HECs, labels weakly in To confirm our finding that 16E5 significantly increases plasma membrane-associated caveolin-1, total cellular membranes were fractionated on sucrose density gradients that separate plasma membrane from organelle membranes (Denker et al, 1996). As shown (Figure 5b), the plasma membrane-associated proteins yes and Tf-R are primarily present in gradient fractions 1-4, while markers for membranes of the Golgi apparatus (b-COP) and ER (calnexin) are restricted to fractions 9-12.…”

“…Total cellular membranes were fractioned on sucrose density gradients according to Denker et al (1996). Detergentresistant membranes were fractionated using a modification of procedures described by Sotgia et al (2002).…”

Possible Involvement of Heterotrimeric G Proteins in the Organization of the Golgi Apparatus

“…Another possibility is that heterotrimeric G proteins on the Golgi apparatus may transduce signals in a different way from that on the plasma membrane. This possibility is raised by the finding that G␤␥ may not be on the Golgi apparatus (45). Since no signals were required for constitutive vesicular transport, heterotrimeric G proteins may be regulated by a mechanism other than by signals formed by the interaction between signal molecules and their receptors.…”

Dynamic Targeting of the Agonist-stimulated m2 Muscarinic Acetylcholine Receptor to Caveolae in Cardiac Myocytes

“…Membrane Markers-Mannosidase II activity was determined by hydrolysis of p-nitrophenyl-␣-D-mannopyranoside (Sigma) with volumes reduced to facilitate the assay in 96-well plates, as described previously (29). After incubation at 37°C for 1 h followed by quenching with 100 mM NaOH, absorbance was measured at 405 nm using a Microplate Reader (SLT Lab Instruments).…”

“…The gradient fractions were also analyzed for their protein content as well as for the presence of mannosidase II, as a Golgi marker (29), and for the level of specific [ 3 H]oubain binding (30), as a specific marker of (Na ϩ , K ϩ )-ATPase, a relatively evenly distributed enzyme at the sarcolemmal surface of cardiac myocytes. As shown by the pattern of distribution of these markers across the gradient (Fig.…”