Protein phosphorylation/dephosphorylation and stimulus-secretion coupling in wild type and mutant Paramecium.
Axenic cultures of Paramecium tetraurelia take up 32Pi and phosphorylate a number of polypeptides as determined by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The most heavily labeled polypeptide has an apparent Mr of approximately 65,000. Wild type cells stimulated to secrete with picric acid, the standard secretagogue for these cells, show a marked reduction in labeling of the 65,000 Mr polypeptide. There is no change in the Coomassic blue staining protein pattern after addition of picric acid. Addition of picric acid to cells solubilized in sample buffer containing 10% sodium dodecyl sulfate, significantly lowers the pH but does not induce dephosphorylation of the 65,000 Mr polypeptide. Dephosphorylation of the 65,000 Mr polypeptide is further correlated with secretion in two types of experiments. 1) Preincubation of cells in Mg2+ (no added Ca2+) inhibits both secretion and dephosphorylation in response to picric acid. 2) A temperature-sensitive mutant, nd 9, when grown at 18 degrees C (permissive temperature) has the normal intramembrane particle array (rosette) at the secretory site and secretes and dephosphorylates the 65,000 Mr polypeptide in response to picric acid, but when grown at 27 degrees C (nonpermissive temperature) does not have assembled rosettes at the secretory site, and does not secrete nor dephosphorylate the 65,000 Mr polypeptide in response to picric acid. This represents the first correlation between a phosphoprotein and a physiological activity (secretion) in Paramecium. Our results show the presence of an in vivo stimulus-sensitive phosphoprotein of Mr 65,000 which appears related to Ca2+-mediated exocytosis. Inhibition of dephosphorylation occurs when secretion is blocked, either by Mg2+ or by a mutation affecting an intramembrane particle array, the rosette.
D000818:Animals; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation; D010247:Paramecium / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000235:genetics*; D013045:Species Specificity; D013696:Temperature
D010750:Phosphoproteins; D011506:Proteins; D008274:Magnesium