Carbohydrate cycling in signal transduction: Parafusin, a phosphoglycoprotein and possible Ca²⁺-dependent transducer molecule in exocytosis in Paramecium

Parafusin, a cytosolic phosphoglycoprotein of M_r 63,000, is dephosphorylated and rephosphorylated rapidly in a Ca²⁺-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°C), does not exocytose or dephosphorylate parafusin upon stimulation in the presence of Ca²⁺; grown at the permissive temperature (18°C), nd9 cells show a wt phenotype. Parafusin contains two types of phosphorylation sites: one where glucose 1-phosphate is added by an α-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′-[β-[³⁵S]thio])diphospho(1)-glucose (UDP[β³⁵S]-Glc) and [γ-³²P]ATP, respectively. The characteristics of these pathways are different. Specifically, in the presence of Ca²⁺, the amount of UDP[β³⁵S]-Glc label in parafusin is reduced. In contrast, identical labeling experiments with [γ-³²P]ATP show that Ca²⁺ enhances labeling of parafusin. Mg²⁺ had no appreciable effect on either labeling. Removal of the UDP[β³⁵S]-Glc label on parafusin in the presence of Ca²⁺ correlates with the in vivo dephosphorylation seen upon exocytosis. Incubations with UDP[β³⁵S]-Glc were then performed with homogenates and nd9 cell fractions grown at 27°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 resides 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 α-glucose 1-phosphate and more generally that carbohydrates on cytoplasmic glycoproteins may be cyclically added and/or removed in response to extracellular stimuli.