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

Sukanya V. Subramanian, Birgit H. Satir

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Abstract

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 languageEnglish (US)
Pages (from-to)11297-11301
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number23
StatePublished - Dec 1 1992

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Paramecium
Exocytosis
Transducers
Signal Transduction
Carbohydrates
Uridine Diphosphate
Phosphoric Diester Hydrolases
Adenosine Triphosphate
Temperature
Uridine Diphosphate Glucose
Uridine
Phosphoric Monoester Hydrolases
Protein Kinases
Serine
Glycoproteins
Phosphotransferases
Phosphates
Phosphorylation
Phenotype

Keywords

  • Ciliates
  • Membrane fusion
  • Secretion

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

@article{9886e344a65a4a6989f924bbab7c3d25,
title = "Carbohydrate cycling in signal transduction: Parafusin, a phosphoglycoprotein and possible Ca2+-dependent transducer molecule in exocytosis in Paramecium",
abstract = "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.",
keywords = "Ciliates, Membrane fusion, Secretion",
author = "Subramanian, {Sukanya V.} and Satir, {Birgit H.}",
year = "1992",
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language = "English (US)",
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pages = "11297--11301",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
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TY - JOUR

T1 - Carbohydrate cycling in signal transduction

T2 - Parafusin, a phosphoglycoprotein and possible Ca2+-dependent transducer molecule in exocytosis in Paramecium

AU - Subramanian, Sukanya V.

AU - Satir, Birgit H.

PY - 1992/12/1

Y1 - 1992/12/1

N2 - 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.

AB - 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.

KW - Ciliates

KW - Membrane fusion

KW - Secretion

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