ALDOSTERONE-INDUCED AND GTP-STIMULATED METHYLATION OF A 90-KDA POLYPEPTIDE IN THE APICAL MEMBRANE OF A(6) EPITHELIA

Aldosterone treatment of A6 cultured renal epithelial cells methylates the apical membrane, and we examined the aldosterone-induced carboxym ethylation of the apical membrane of these cells to determine the targ eted polypeptides. Methionine-deprived A6 cells were incubated with al dosterone and [H-3]methionine. Homogenates and apical membranes were s olubilized and analyzed by SDS-polyacrylamide gel electrophoresis. Lab el incorporation in a 90-kDa polypeptide was more intense (4-fold) in membranes after aldosterone compared to control. For in vitro methylat ion, membranes were isolated, incubated with S-adenosyl-L-[methyl-H-3] methionine, and analyzed for H-3-methyl uptake. Label incorporation wa s low in control membranes but markedly stimulated (4-fold) in membran e preparations from aldosterone-treated cells. Guanosine 5'-O-(3-thiot riphosphate) increased in vitro methylation of a 90-kDa polypeptide 5- fold in control membranes but after aldosterone, where methylation was already stimulated, little change was observed. We conclude that aldo sterone induces methylation of an apical membrane 90-kDa polypeptide, possibly a subunit of the epithelial Na+ channel, in a GTP-dependent m anner, and this may be one of the final steps in a cascade of reaction s leading to the natriferic action of this hormone.