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 ...