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

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 alpha subunit of Gs (Gs alpha) Ransn?s, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900-7903], three membrane-bound forms of Gs alpha were identified through rate-zonal centrifugation in sucrose density gradients, Gs alpha-specific anti-peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gs alpha in the high-density portion of the gradient (1.13-1.16 g/cm3) is identical with that of beta-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 Gs alpha and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH-cytochrome-c reductase and glucose-6-phosphatase. The identity of the third pool of Gs alpha 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 Gs alpha is distinct from any plasma-membrane fragments. Furthermore, sedimentation at 100,000 x g proves its particulate (membrane) character. The light membrane form of Gs alpha subunit is functionally active in an adenylate cyclase complementation assay using cyc- membranes devoid of Gs alpha. Overall, our data indicates that a substantial portion of Gs alpha is localized in membrane pools other than plasma membrane.