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) [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 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.
Svoboda
P
P
Institute of Physiology, Czech Academy of Sciences, Prague.
Kvapil
P
P
Insel
P A
PA
Ransnäs
L A
LA
D000262:Adenylyl Cyclases / Q000378:metabolism; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008223:Lymphoma; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D000254:Sodium-Potassium-Exchanging ATPase; D005459:Fluorides