Open AccessPlasma‐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
Author(s) -
SVOBODA Petr,
KVAPIL Petr,
INSEL Paul A.,
RANSNÄS Lennart A.
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb17236.x
Subject(s) - endoplasmic reticulum , adenylate kinase , cyclase , differential centrifugation , g alpha subunit , biochemistry , phosphatase , microbiology and biotechnology , biology , atpase , golgi apparatus , phosphotransferase , gs alpha subunit , protein subunit , chemistry , g protein , enzyme , receptor , gene
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 α subunit of G s (G s α) [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 G s α were identified through ratezonal centrifugation in sucrose density gradients, G s α‐specific anti‐peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of G s α in the high‐density portion of the gradient (1.13–1.16 g/cm 3 ) 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/cm 3 ), contains a much lower total amount of G s α and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH–cytochrome‐ c reductase and glucose‐6‐phosphatase. The identity of the third pool of G s α located at the top of the gradient (1.06–1.08 g/cm 3 ), 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 α is distinct from any plasma‐membrane fragments. Furthermore, sedimentation at 1 × g proves its particulate (membrane) character. The light membrane form of G s α subunit is functionally active in an adenylate cyclase complementation assay using cyc − membranes devoid of G s α. Overall, our data indicates that a substantial portion of G s α is localized in membrane pools other than plasma membrane.