title: Carbohydrate cycling in signal transduction: parafusin, a phosphoglycoprotein and possible Ca(2+)-dependent transducer molecule in exocytosis in Paramecium.
abstract: Parafusin, a cytosolic phosphoglycoprotein of M(r) 63,000, is dephosphorylated and rephosphorylated rapidly in a Ca(2+)-dependent manner upon stimulation of exocytosis in vivo in wild-type (wt) Paramecium. In contrast, the temperature-sensitive exocytosis mutant nd9, grown at the nonpermissive temperature (27 degrees C), does not exocytose or dephosphorylate parafusin upon stimulation in the presence of Ca2+; grown at the permissive temperature (18 degrees C), nd9 cells show a wt phenotype. Parafusin contains two types of phosphorylation sites: one where glucose 1-phosphate is added by an alpha-glucose-1-phosphate phosphotransferase and removed by a phosphodiesterase and one where phosphate from ATP is added directly to a serine residue by a protein kinase and removed by a phosphatase. We show here that, in cell fractions from wt Paramecium, both reactions can be carried out in vitro by using uridine(5'-[beta-[35S]thio])diphospho(1)-glucose (UDP[beta 35S]-Glc) and [gamma-32P]ATP, respectively. The characteristics of these pathways are different. Specifically, in the presence of Ca2+, the amount of UDP[beta 35S]-Glc label in parafusin is reduced. In contrast, identical labeling experiments with [gamma-32P]ATP show that Ca2+ enhances labeling of parafusin. Mg2+ had no appreciable effect on either labeling. Removal of the UDP[beta 35S]-Glc label on parafusin in the presence of Ca2+ correlates with the in vivo dephosphorylation seen upon exocytosis. Incubations with UDP[beta 35S]-Glc were then performed with homogenates and nd9 cell fractions grown at 27 degrees C under the ionic conditions used for wt cells. These labelings were not affected by Ca2+, in contrast to results from wt cells but in accord with those obtained earlier with nd9-27 mutant cells in vivo. Factors responsible for both dephosphorylation and Ca2+ sensitivity were found in the high-speed pellet (P2) in wt cells, suggesting that the putative phosphodiesterase is in this fraction and that the defect in the mutant nd9-27 residues in the Ca2+ activation of the phosphodiesterase. We conclude that the in vivo dephosphorylation of parafusin that occurs upon exocytosis is a dephosphoglucosylation due to removal of the alpha-glucose 1-phosphate and more generally that carbohydrates on cytoplasmic glycoproteins may be cyclically added and/or removed in response to extracellular stimuli.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY 10461.
authors.forename: S V
authors.initials: SV
authors.lastname: Subramanian
authors.forename: B H
authors.initials: BH
authors.lastname: Satir
mesh_terms: D002118:Calcium / Q000502:physiology; D050260:Carbohydrate Metabolism; D004789:Enzyme Activation; D005089:Exocytosis; D005964:Glucosyltransferases / Q000378:metabolism; D006001:Glycoconjugates / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D006001:Glycoconjugates; D006023:Glycoproteins; D010750:Phosphoproteins; D015800:Protozoan Proteins; D005964:Glucosyltransferases; D010770:Phosphotransferases; D010727:Phosphoric Diester Hydrolases; D002118:Calcium