Enzymatic Regulation of Membrane Fusion During Synchronous Exocytosis in Paramecium Cells

Abstract

We have used Paramecium tetraurelia cells to analyze parameters relevant for membrane fusion regulation during exocytosis for different reasons: This cell type secretes trichocysts synchronously (1 sec); there exist secretory mutants; finally, the cell cortex can be isolated and used to analyze exocytosis in vitro. We have found with SDS-FAGE-ARG from 32P-prelabeled cells that the selective dephosphorylation of a 65 kD PP always parallels exocytosis. Its extent depends on the amount of trichocysts actually released (dose-dependence in “normal” cells; none in non-discharge mutations; variable in cells during replenishment of the trichocyst pool after a preceding trigger). SDS-PAGE-ARG or immuno labeling reveal 65 kD PP in the cell cortex, which also contains CaM (localized by affinity- or immuno-labeling specifically on the preformed exocytosis sites) and a CaN-like protein (shown on nitrocellulose blots). Ca+CaM+CaN stimulates exocytosis in vitro, as it does, after microinjection, in vivo. (Alkaline phosphatase can mimic this effect). Concomitantly, αCaM- or αCaN-AB suppress exocytosis. We could also show that Ca+CaM+CaN causes 65 kD PP dephosphorylation during exocytosis. The Ca2+-CaM triggered dephosphorylation of the 65 kD PP, mediated by a CaN-type phosphatase, is therefore considered as the/a primary step in fusion regulation during exocytosis. ATP possibly reduces the fusion capacity, at least in part, via protein rephosphorylation. In our system we could also ascertain that ATP consumption as well as protein phosphorylation occur only ≳5 seconds after synchronous exocytosis and protein dephosphorylation. The synchronicity of our system allows to avoid any possible overlaps of these phenomena.