Synchronous exocytosis in Paramecium cells involves very rapid (less than or equal to 1 s), reversible dephosphorylation of a 65-kD phosphoprotein in exocytosis-competent strains.

Zieseniss, Egmont; Plattner, Helmut

doi:10.1083/jcb.101.6.2028

Cited by 83 publications

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“…Manual pipetting allows recognition of PP63 dephosphorylation only in the range of 1 s and to a smaller extent (Zieseniss & Plattner, 1985 and conservation of the dephosphorylated state was another approach (Gilligan & Satir, 1982). Any other analyses reported in the literature were conducted on a broader time scale than that analysed here.…”

Section: Discussion Time Course Of Pp63 Dephosphorylationmentioning

confidence: 99%

“…2a). Quantitative evaluation of the 63 kDa band by density scans showed partial (Zieseniss & Plattner, 1985).…”

Section: Rapid Dephosphorylation Of Pp63mentioning

confidence: 96%

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mentioning

confidence: 99%

“…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).…”

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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: Discussion Time Course Of Pp63 Dephosphorylationmentioning

confidence: 99%

“…2a). Quantitative evaluation of the 63 kDa band by density scans showed partial (Zieseniss & Plattner, 1985).…”

Section: Rapid Dephosphorylation Of Pp63mentioning

confidence: 96%

“…

…”

mentioning

confidence: 99%

mentioning

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 have employed this strategy in investigating phosphorylation sites in the 63 kDa phosphoprotein PP63/ parafusin (PP63/pf) 1 of the ciliated protozoan Paramecium tetraurelia (6,7). This protein is heavily enriched in the cell cortex (8).…”

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confidence: 99%

Comparison of in Vivo and in Vitro Phosphorylation of the Exocytosis-Sensitive Protein PP63/Parafusin by Differential MALDI Mass Spectrometric Peptide Mapping

et al. 1999

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PP63 (parafusin) is a 63 kDa phosphoprotein, which exists in at least two different isoforms. It is very rapidly (80 ms) dephosphorylated during triggered trichocyst exocytosis. This occurs selectively in exocytosis-competent Paramecium tetraurelia strains. At least two protein kinases isolated from Paramecium, casein kinase type II kinase and cGMP-dependent kinase, are able to phosphorylate the two recombinant PP63/parafusin isoforms, both with phosphoglucomutase activity, in vitro. By performing mass spectrometric peptide mapping, we have investigated in vitro phosphorylation of recombinant PP63/ parafusin by these kinases in comparison to in vivo phosphorylation of native PP63/parafusin isolated from Paramecium homogenates. Low picomolar quantities of proteolytic digests of recombinant and native PP63/parafusin, prior to and following alkaline phosphatase treatment, were directly analyzed by MALDI mass spectrometry. In native PP63-1/parafusin-1, six of 64 serine and threonine residues (S-196, T-205, T-280, T-371, T-373, and T-469) were found definitely, 27 were found possibly phosphorylated, 28 were identified as nonphosphorylated, and three were not covered by mapping. Three of the six certainly phosphorylated amino acids represent consensus phosphorylation sites for casein kinase II or cGMPdependent protein kinase. In vitro phosphorylation studies of recombinant PP63/parafusin confirm that some of the sites found were used in vivo; however, also significant differences with respect to in vivo phosphorylation of native PP63/parafusin were observed. The two Paramecium protein kinases that were used do not preferably phosphorylate expected consensus sites in vitro. Homology structure modeling of PP63/parafusin with rabbit phosphoglucomutase revealed that the majority of residues found phosphorylated is located on the surface of the molecule.

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Membrane Fusion

Blumenthal

Dimitrov

1997

Comprehensive Physiology

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The sections in this article are: Observation of Fusion Requires Physical Techniques for Monitoring Mixing of Membranes and the Compartments they Enclose Morphological Changes Following Fusion Are Observed by Light Microscopy but Membrane Fusion May Occur without such Changes Fluorescence Microscopy and Spectrofluorometry Allow Quantitation of Membrane Fusion Events in Living Cells Electron Microscopy Provides Direct Observation of Structural Rearrangements Due to Fusion Patch‐Clamp Techniques Allow the Monitoring of Very Fast Openings of Fusion Pores What Do We Learn from “Nonbiological” Fusion Processes? Ca 2+ Induces Aggregation Destabilization, and Fusion of Liposomes Containing Phospholipids with Negatively Charged Head‐groups Fusion of Lipid Membranes by Amphipathic and Nonpolar Molecules Correlates with Their Lytic and Aggregational Activity Dehydration, Aggregation, and Destabilization of Membranes by Polyethelene Glycol Are Essential for Fusion of Lipid Membranes Destabilization by High‐Voltage Electric Pulses Leads to Fusion of Adjoining Membranes Molecular Rearrangements in the Lipid Bilayers during the Very Act of Fusion May Involve Intermediate Structures Specialized Proteins Mediate Fusion in Life Processes Viral Envelope Proteins Contain Hydrophobic “Fusion Peptide” Sequences To Enter a Cell a Virus Must Find the Receptor That Invites It In Some Viruses Require More Than One Type of Envelope Protein for Entry Influenza Hemagglutinin Was the Only Fusion Protein with Known Three‐Dimensional Structure The Process of HA ‐mediated Membrane Fusion Can Be Dissected into a Number of Elementary Steps Human Immunodeficiency Virus Type 1 ( HIV ‐1), the Primary Etiological Agent of the Acquired Immunodeficiency Syndrome ( AIDS ), Enters Cells by Membrane Fusion at Neutral pH The Receptor CD 4 Plays Both a Passive and an Active Role in Allowing Entry of the Virus into the Cell Stable Envelope Glycoprotein‐Receptor Complex Formation Is Rate‐limiting in the Overall Fusion Process Multiple Copies of the HIV ‐1 Envelope Glycoprotein May Be Required for Fusion Pore Formation Sperm Membrane Proteins Involved in Sperm‐Egg Fusion May Resemble Viral Fusion Proteins Toward A Resolution of Fusion Proteins in Exocytosis Multiple Proteins May Be Required for Intracellular Fusion Toward A Physicochemical Analysis of Fusion Kinetics Delays in Fusion Are Proportional to the Fusion Barriers and Decrease with an Increase in the Strength of the Fusogen Rates of Fusion Can Provide Information for the Time Course of Membrane Merging and Fusion Pore Expansion Fusion Yields and Delays Are Related but May Reflect Different Properties of the Fusing Membranes Does Understanding Membrane Fusion Need New Breakthroughs in Methodology? Note Added in Proof

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Synchronous exocytosis in Paramecium cells involves very rapid (less than or equal to 1 s), reversible dephosphorylation of a 65-kD phosphoprotein in exocytosis-competent strains.

Cited by 83 publications

References 37 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

Comparison of in Vivo and in Vitro Phosphorylation of the Exocytosis-Sensitive Protein PP63/Parafusin by Differential MALDI Mass Spectrometric Peptide Mapping

Membrane Fusion

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