Parafusin, an exocytic-sensitive phosphoprotein, is the primary acceptor for the glucosylphosphotransferase in Paramecium tetraurelia and rat liver.

Satir, Birgit H.; Srisomsap, Chantragan; Reichman, Milaniya; Marchase, Richard B.

doi:10.1083/jcb.111.3.901

Cited by 34 publications

(29 citation statements)

References 29 publications

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“…Previous indications on seryl-(or threonyl-) type phosphorylation sites (Murtaugh et al, 1987) may be valid, as we also found them to be sensitive to alkaline, but insensitive to acid, hydrolysis. [It was not analysed in any detail why (P)P63 forms of different pI values occur, and whether one form is glycosylated, as suggested by Satir et al (1990). ] For all these reasons we used the pI-5.95 form of P63 and of PP63 for immunolocalization studies.…”

Section: Isoforms Of Pp63mentioning

confidence: 99%

A cortical phosphoprotein (‘PP63’) sensitive to exocytosis triggering in Paramecium cells. Immunolocalization and quenched-flow correlation of time course of dephosphorylation with membrane fusion

Höhne-Zell

Knoll

Riedel-Gras

et al. 1992

Biochemical Journal

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We had previously shown that a phosphoprotein of 63 kDa ('PP63') is rapidly and selectively dephosphorylated during synchronous (< 1 s) trichocyst exocytosis in Paramecium cells and then rephosphorylated within < 1 min [Zieseniss & Plattner (1985) J. Cell Biol. 101, 2028-2035. Using a new quenched-flow device, we now find a strict correlation between PP63 dephosphorylation and the process of membrane fusion, both occurring within 80 ms. Uptake of 32P over 90 min, followed by exocytosis and rephosphorylation for 1 min, results in a rather selective phosphorylation of the dephosphorylated form, P63, to PP63. Solubilization by repeated freezing and thawing allows isolations of P63 and PP63. On isoelectric focusing autoradiograms they have pl values of 6.05, 5.95 (major spots), 5.85 and 5.75. All spots are sensitive to alkaline, but not to acidic, hydrolysis (except for the pl-6.05 spot). On two-dimensional-gel autoradiograms the most prominent spot, of pl 5.95, is most extensively de-and re-phosphorylated. This spot, from de-and rephosphorylated samples, was used to produce monospecific antibodies. A cortical localization of PP63 was revealed by producing Western blots from isolated cell-surface fragments ('cortices') and by immunofluorescence labelling. We assume that both P63 and PP63 are attached to cortical structures, e.g. around trichocysts, though they are partly soluble. This localization and the strict correlation of PP63 dephosphorylation with exocytotic membrane fusion suggests a role in fusion regulation. INTRODUCTION MATERIALS AND METHODSThough a change in the degree of phosphorylation of different proteins is a key event accompanying triggered secretory activity in many cells, no phosphoprotein (PP) has so far been shown to be directly involved in the regulation of vesicle-cell-membrane interaction for fusion (for reviews, see Hemmings et al., 1989;Plattner, 1989;Burgoyne, 1991). Some of the uncertainties come from superposition of different steps, such as organelle docking, membrane fusion, resealing and internalization. A synchronous system allowing for the selective analysis of specific steps would be of evident advantage. This is the case with Paramecium cells, whose trichocysts can be released synchronously upon triggering with certain polyaminated secretagogues . The recent development of a quenched-flow procedure applicable to such fragile cells now allows for simultaneous analysis by ultrastructural and biochemical methods, specifically of the membrane fusion process. The present investigation was focused on the dephosphorylation of a PP of 63 kDa (PP63, formerly 65 kDa), a salient feature of trichocyst exocytosis (Gilligan & Satir, 1982;Zieseniss & Plattner, 1985). We analysed the precise time course of PP63 dephosphorylation, the occurrence of PP63 isoforms of different pl values and their sensitivity to exocytosis triggering, as well as the subcellular localization of PP63 and its dephosphorylated form (P63). We prepared monospecific antibodies for Western blots and for immunocytochemical ...

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Section: Isoforms Of Pp63mentioning

confidence: 99%

A cortical phosphoprotein (‘PP63’) sensitive to exocytosis triggering in Paramecium cells. Immunolocalization and quenched-flow correlation of time course of dephosphorylation with membrane fusion

Höhne-Zell

Knoll

Riedel-Gras

et al. 1992

Biochemical Journal

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“…In this reaction, Glc-1-P is transferred from UDP-Glc to one or more mannose residues on phosphoglucomutase through the action of a membrane-bound Glc-phosphotransferase. While the purpose of this posttranslational modification of phosphoglucomutase is not yet understood, its occurrence in organisms as diverse as S. cerevisiae, Paramecium tetraurelia (26), and mammals (32) suggests that it carries out an important, conserved function. To better understand the function of the posttranslational modification of phosphoglucomutase in yeast cells, we have compared the abundance of phosphoglucomutase, its enzymatic activity, and its posttranslational modification under different physiological conditions.…”

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The posttranslational modification of phosphoglucomutase is regulated by galactose induction and glucose repression in Saccharomyces cerevisiae

Bounelis

Dey

et al. 1995

J Bacteriol

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The enzyme phosphoglucomutase functions at a key point in carbohydrate metabolism. In this paper, we show that the synthesis of the major isoform of yeast phosphoglucomutase, encoded by the GAL5 (PGM2) gene, is regulated in a manner that is distinct from that previously described for other enzymes involved in galactose metabolism in the yeast Saccharomyces cerevisiae. Accumulation of this isoform increased four-to sixfold when the culture experienced either glucose depletion or heat shock. However, heat shock induction did not occur unless the cells were under glucose repression. This nonadditive increase in expression suggests that the regulatory mechanisms controlling the heat shock induction and glucose repression of the GAL5 gene are functionally related. We previously demonstrated that phosphoglucomutase is modified by a posttranslational Glc-phosphorylation reaction. We now show that this posttranslational modification, like phosphoglucomutase expression itself, is also regulated by galactose induction and glucose repression. Finally, no evidence was found to indicate that the Glc-phosphorylation of phosphoglucomutase alters its enzymatic activity under the conditions examined.The interconversion of Glc-1-P and Glc-6-P, a key metabolic trafficking point in all cells, is carried out by the enzyme phosphoglucomutase (EC 2.7.5.1). In Saccharomyces cerevisiae cells grown on galactose as the primary carbon source, the Glc-1-P formed as a result of galactose metabolism is converted to Glc-6-P by phosphoglucomutase as a required step in providing cells with carbon-containing precursors. When yeast cells are grown on other carbon sources, phosphoglucomutase functions primarily to convert Glc-6-P to Glc-1-P. Glc-1-P is required for the synthesis of the sugar nucleotide UDP-Glc, which acts as a precursor for the synthesis of oligosaccharides and trehalose. Phosphoglucomutase consists of two isoforms in S. cerevisiae (31). The major isoform is encoded by the GAL5 (PGM2) gene (8), while the gene encoding the minor isoform is called PGM1 (3).The synthesis of most enzymes involved in the galactose utilization (Leloir) pathway is subject to tight control by overlapping mechanisms of glucose repression and galactose induction, leading to as much as a 1,000-fold induction of enzyme levels when a yeast culture is shifted from glucose to galactose medium. In contrast, phosphoglucomutase was regarded as a constitutively expressed enzyme for many years (3, 12). Since phosphoglucomutase function is also required for growth on media containing glucose as the carbon source, this conclusion was consistent with its metabolic function. However, phosphoglucomutase synthesis is also regulated by glucose repression and galactose induction, although the magnitude of this regulation is considerably weaker than that of other genes involved in galactose metabolism (23). The galactose regulation of the GAL5 gene, like other genes encoding proteins in the Leloir pathway, is mediated by the Gal4p-Gal80p regulatory system. Gal4p acts as a posi...

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“…§1734 solely to indicate this fact. et al (10) have determined that in Paramecium aGlc-1-P is similarly added to mannose residues in parafusin by an aGlc-1-P phosphotransferase and that parafusin from Paramecium and the liver phosphoglycoprotein have the same major characteristics.…”

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Carbohydrate cycling in signal transduction: parafusin, a phosphoglycoprotein and possible Ca(2+)-dependent transducer molecule in exocytosis in Paramecium.

Subramanian

Satir

1992

Proc. Natl. Acad. Sci. U.S.A.

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Parafusin, a cytosolic phosphoglycoprotein of The background data that tie parafusin to the process of membrane fusion and exocytosis come from studies using the ciliates Paramecium tetraurelia and Tetrahymena thermophila. Paramecium tetraurelia in axenic cultures take up 32p, and phosphorylate a number of polypeptides. The most heavily labeled polypeptide is parafusin, a minor component of Mr 63,000 that shows serine phosphorylation (1, 2). Gilligan and Satir (3,4) showed that, when in vivo-prelabeled wild-type (wt) cells were stimulated to secrete the content of their docked dense-core secretory vesicles (trichocysts) with trinitrophenol, the labeled parafusin was dramatically dephosphorylated. Dephosphorylation was correlated with the massive exocytosis of secretory vesicles in two types of experiments: one testing ionic requirements for exocytosis (Ca2+ vs. Mg2+) and the other using exocytosis mutants.Exocytosis and dephosphorylation required the presence of Ca2+ in the medium in that 5 mM MgCl2, in the absence of added Ca2+, inhibited both processes. Both membrane fusion and changes within the secretory vesicle content are Ca2+-dependent processes, the former requiring an increase of cytosolic Ca2+, probably via receptor-gated Ca2+ channels in the membrane (5). Further, the temperature-sensitive secretory mutant nd9 grown at the restrictive temperature (270C) and then stimulated with trinitrophenol in the presence of 5 mM Ca2+ neither exocytosed nor dephosphorylated parafusin (3). Dephosphorylation and exocytosis, very rapid events, are normally followed by rephosphorylation within 10 sec (6).Parafusin was isolated and purified to apparent homogeneity. An affinity-purified polyclonal antibody was made that recognizes both the phosphorylated and dephosphorylated forms. Two slightly acidic (pI = 5.8 and 6.2) phosphorylated forms of parafusin are found in a cytosolic fraction. The affinity-purified antibody recognizes a third isoelectric form at pI 6.3 that appears unlabeled and is probably membrane bound (1, 2). Parafusin shows immunological cross-reactivity with proteins from a variety ofunicellular organisms and cells of metazoan groups of wide evolutionary divergence, as well as with rat tissues, including brain, heart, pituitary, and kidney. These results suggest that parafusin was present early in the history of eukaryotes and may be of functional importance in the general mechanism of exocytosis and membrane fusion (7,8).A Mr 62,000 phosphoglycoprotein in liver that possessed an a-glucose-l-phosphate (aGlc-l-P) moiety linked to a short chain of mannoses that was O-linked to a senine residue of the protein was independently discovered (9). The bond between glucose and mannose in this protein was a phosphodiester linkage formed by the action of a unique aGlc-1-P phosphotransferase (9). By incorporation of label from a thiophos-

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Parafusin, an exocytic-sensitive phosphoprotein, is the primary acceptor for the glucosylphosphotransferase in Paramecium tetraurelia and rat liver.

Cited by 34 publications

References 29 publications

A cortical phosphoprotein (‘PP63’) sensitive to exocytosis triggering in Paramecium cells. Immunolocalization and quenched-flow correlation of time course of dephosphorylation with membrane fusion

A cortical phosphoprotein (‘PP63’) sensitive to exocytosis triggering in Paramecium cells. Immunolocalization and quenched-flow correlation of time course of dephosphorylation with membrane fusion

The posttranslational modification of phosphoglucomutase is regulated by galactose induction and glucose repression in Saccharomyces cerevisiae

Carbohydrate cycling in signal transduction: parafusin, a phosphoglycoprotein and possible Ca(2+)-dependent transducer molecule in exocytosis in Paramecium.

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