Species distribution of a phosphoprotein (parafusin) involved in exocytosis.

Satir, Birgit H.; Hamasaki, Toshikazu; Reichman, Milaniya; Murtaugh, Timothy J.

doi:10.1073/pnas.86.3.930

Cited by 35 publications

(21 citation statements)

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“…During two-dimensional gel electrophoresis and autoradiography we consistently lost the first spot during SDS/ PAGE. Interestingly, the 32P label also was completely lost, probably for the same reason, with the isolation protocol used by Satir et al (1989), although the antibodies that they prepared against this form recognize the phosphorylated 63 kDa band on blots from SDS/PAGE. Altogether, their data may be difficult to compare with ours, since the antibodies that we prepared were against the well-defined spot of pI 5.95 of 63 kDa.…”

Section: Isoforms Of Pp63mentioning

confidence: 99%

“…Since a phosphatase system is located in the cell cortex , this might account for the loss Of 32P label from cortices and for the selective detectability of PP63 in supernatants by gel autoradiography, whereas detection by antibodies is less ambiguous. Satir et al (1989) suggested the occurrence of a Ca2+_ dependent sedimentable pool which had previously remained undetected. Yet we found no indications of Ca2+ binding by P63 or PP63 (see the Results section).…”

Section: Localization Of P63/pp63mentioning

confidence: 99%

“…Satir et al (1989) have reported pl values of 6.2 and 5.8. During two-dimensional gel electrophoresis and autoradiography we consistently lost the first spot during SDS/ PAGE.…”

Section: Isoforms Of Pp63mentioning

confidence: 99%

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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%

Section: Localization Of P63/pp63mentioning

confidence: 99%

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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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“…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).…”

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confidence: 99%

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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“…The coincidence of exocytosis competence and pp63/pf N de-phosphorylation led to the proposal that this protein may govern membrane fusion, from which the name was derived [30][31][32]. Cloning of its homologue in Tetrahymena and disruption of its single pp63/pf gene suggested this not to be the case [33].…”

Section: Introductionmentioning

confidence: 99%

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells

Plattner

Kissmehl

2005

Cell Calcium

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Calf signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the ::s10 3 dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Calf -dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-Iocalised, and characterised as phosphoglucomutase. the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo. followed by MALDl analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial SerlThr residues. Although present also in exo-mutants. pp63/pf is selectively de-phosphorylated only in exo+ strains during synchronous exocytosis (80 ms) and re-phosphorylated within~20 s, Le., the time required to re-establish [Calf] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider CalfIcalmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Calf-activated guanylate cyclase. Another kinase, casein kinase 2. is inhibited by Calf and. hence, activated with some delay in parallel to decreasing [Calf] after exocytosis. In total, several Calf -sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo-mutants) and NADH formation. with effects on Calf signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca 2 + dynamics during synchronous exo-and endocytosis.

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Species distribution of a phosphoprotein (parafusin) involved in exocytosis.

Cited by 35 publications

References 9 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

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

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells

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