Parafusin, a cytosolic phosphoglycoprotein of Mr 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.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Publication status||Published - Dec 1 1992|
- Membrane fusion
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