Plasma‐membrane‐independent pool of the α subunit of the stimulatory guanine‐nucleotide‐binding regulatory protein in a low‐density‐membrane fraction of S49 lymphoma cells

Abstract

We report that compartmentalisation of the stimulatory guanine‐nucleotide‐binding regulatory protein (Gs) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the α subunit of Gs (Gsα) [Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900–7903], three membrane‐bound forms of Gsα were identified through ratezonal centrifugation in sucrose density gradients, Gsα‐specific anti‐peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gsα in the high‐density portion of the gradient (1.13–1.16 g/cm3) is identical with that of β‐adrenergic‐receptor binding, Na/K‐ATPase and adenylate cyclase activity, and may therefore be identified as plasma‐membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09–1.11 g/cm3), contains a much lower total amount of Gsα and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH–cytochrome‐c reductase and glucose‐6‐phosphatase. The identity of the third pool of Gsα located at the top of the gradient (1.06–1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose:N‐acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high‐density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K‐ATPase activity indicates that this low‐density (light) membrane form of Gsα is distinct from any plasma‐membrane fragments. Furthermore, sedimentation at 100000 ×g proves its particulate (membrane) character. The light membrane form of Gsα subunit is functionally active in an adenylate cyclase complementation assay using cyc− membranes devoid of Gsα. Overall, our data indicates that a substantial portion of Gsα is localized in membrane pools other than plasma membrane.

Purification of Heterotrimeric G Protein α Subunits by GST-Ric-8 Association

“…GST-Ric-8 proteins are mostly cytosolic, whereas functional endogenously expressed G proteins are viewed to reside predominantly on the plasma membrane with only a small subfraction present in the cytosol. Studies have shown that a population of some G proteins becomes released from the plasma membrane into a soluble fraction upon GPCR agonist treatment (42)(43)(44)(45). Perhaps a function of Ric-8 is to bind the G␣ released into the cytosol and recycle it back to the plasma membrane.…”

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

“…We were able to rule out that the presence of Get subunits in Golgi fractions resuits from contamination by PM because the depletion of >95 % of PM by WGA lectin absorption of Golgi fractions did not deplete G%, Getq/ll , or Goti3. Goti3 has previously been localized to Golgi cisternae by immunoelectron microscopy (54,60), but the distribution of different families of Get proteins among Golgi elements has not been investigated up to now.…”

The decrease in the short variant of gsalpha protein is associated with an increase in [3H]CGP12177 binding, [3H]ouabain binding and Na, K-ATPase activity in brown adipose tissue plasma membranes of cold-acclimated hamsters

“…Throughout the years, we have tried to improve this isolation procedure. Based on experience obtained from subcellular fractionation of S49 lymphoma cells (Svoboda et al 1992), the new method for isolation of plasma membrane from BAT was introduced (Svoboda et al 1993). This method used non-equilibrium centrifugation on discontinuous gradients composed of sucrose solutions of exactly defined volumes and densities.…”