IMMUNOELECTRON MICROSCOPIC IDENTIFICATION OF CYTOPLASMIC AND NUCLEAR G(S-ALPHA) IN S49 LYMPHOMA-CELLS

The subcellular distribution of G(s alpha) was characterized in S49 lymphoma cells with two polyclonal antisera directed against specific COOH- and NH2-terminal epitopes. Nonspecific binding was determined in each subcellular compartment by incubating cyc(-) S49 cells, known to be deficient in G(s alpha) and its mRNA, with primary and secondary antisera. Small proportions of total specific binding sites were localized to the plasmalemma as well as the nuclear envelope. Because of their small size, these compartments contained a high concentration of G(s alpha). However, most of the specific binding sites were found in nonstructured cytoplasm and within the nucleus. Specific binding was abolished or significantly reduced by preincubating primary antisera with their peptide immunogens but not with an irrelevant peptide. Intracellular G(s alpha) immunoreactive binding sites did not colocalize with gold-conjugated transferrin in cells preincubated with this ligand to mark a classical endocytotic pathway. The intracellular and intranuclear location of G(s alpha) was confirmed with confocal microscopy of S49 cells immunostained with specific primary and fluorescently labeled secondary antibodies. G(s alpha) was also detected with immunoblots of proteins extracted from purified S49 cell. nuclei. Thus, G(s alpha) is abundantly distributed in intracellular and intranuclear sites in S49 cells and occurs in loci distinct from organelles of the transferrin pathway. The substantial intracellular distribution of G(s alpha) suggests that G(s) may subserve intracellular and, perhaps, intranuclear functions that may be important in proliferating cells.