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

Abstract

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 ...

A novel, calcium‐inhibitable casein kinase in Paramecium cells

“…In some cases, ATP was replaced by 120 uM GTP including [y-32 P]GTP (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Reactions were started by adding protein kinase samples and, after 20 min at 20°C, terminated and measured by spotting 20 ul aliquots either onto trichloroacetic acid-prepared Whatman 1 Chr 3 MM filter papers [42] or, in the case of phosphate incorporation into peptide, by binding to phosphocellulose paper (Whatman P81) as described previously [44].…”

“…Exocytosis capacity is strictly paralleled by the capacity to dephosphorylate a 63 kDa phosphoprotein (PP63), followed by rapid rephosphorylation on Ser/Thr residues [24,25]. A casein kinase analyzed in detail in the present paper phosphorylates PP63 in vitro [26] while dephosphorylation was catalyzed by calcineurin (CaN) [26,27], a protein Ser/Thr phosphatase requiring Ca 2+ and calmodulin (CaM).…”

“…Standard assays for protein kinases were performed in a volume of 0.03 ml containing 5 mM MgCl 2 , 120 uM ATP, [y-32 P]ATP (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) MBq/umol), 19.8 ug mixed casein and buffer (20 mM triethanolamine-HCl buffer, 10% glycerol, 1 mM DTE, pH 7.5) with or without Ca 2+ as indicated. In some cases, ATP was replaced by 120 uM GTP including [y-32 P]GTP (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

“…In Paramecium protein phosphorylation/dephosphorylation cycles are involved in exocytosis regulation [24][25][26]. Exocytosis capacity is strictly paralleled by the capacity to dephosphorylate a 63 kDa phosphoprotein (PP63), followed by rapid rephosphorylation on Ser/Thr residues [24,25].…”

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

“…This protein is heavily enriched in the cell cortex (8). It is selectively dephosphorylated within 80 ms during stimulated synchronous exocytosis of trichocysts and fully rephosphorylated within 10 s (6,9). It is proposed to be involved in the regulation of the exo-and endocytosis cycle since endocytosis rapidly occurs upon phosphorylation of PP63/pf (10).…”