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 (G(s)) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the alpha subunit of G(s) (G(s)alpha) [Ransnas, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900 - 7903], three membrane-bound forms of G(s)alpha were identified through rate-zonal centrifugation in sucrose density gradients, G(s)alpha-specific anti-peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of G(s)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 G(s)alpha and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NA DPH - cytochrome-c reductase and glucose-6-phosphatase. The identity of the third pool of G(s)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 G(s)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 G(s)alpha subunit is functionally active in an adenylate cyclase complementation assay using cyc- membranes devoid of G(s)alpha. Overall, our data indicates that a substantial portion of G(s)alpha is localized in membrane pools other than plasma membrane.