ATP keeps exocytosis sites in a primed state but is not required for membrane fusion : an analysis with Paramecium cells in vivo and in vitro

We have tried to specify a widespread hypothesis on the requirement of ATP for exocytosis (membrane fusion). With Paramecium tetraurelia cells, synchronously (~1 s) exocytosing trichoeysts, ATP pools have been measured in different strains, including wild type cells, "non-discharge" (nd), "trichless" (tl), and other mutations. The occurrence of a considerable and rapid ATP consumption also in nd and tl mutations as well as its time course (with a maximum 3-5 s after exocytosis) in exocytosis-competent strains does not match the actual extent of exocytosis perforrnance. However, from in vivo as well as from in vitro experiments, we came to the conclusion that ATP might be required to keep the system in a primed state and its removal might facilitate membrane fusion. (For the study of exoeytosis in vitro we have developed a new system, consisting of isolated cortices). In vivo as well as in vitro exocytosis is inhibited by increased levels of ATP or by a nonhydrolyzable ATP analogue. In vitro exocytosis is facilitated in ATP-free media. In vivo-microinjected ATP retards exocytosis in response to chemical triggers, whereas microinjected apyrase triggers exocytosis without exogenous trigger. Experiments with this system also largely exclude any overlaps with other processes that normally accompany exoeytosis. Our data also explain why it was frequently assumed that ATP would be required for exocytosis. We conclude (a) that membrane fusion during exocytosis does not require the presence of ATP; (b) the occurrence of membrane fusion might involve the elimination of ATP from primed fusogenic sites; (c) most of the ATP consumption measured in the course of exocytosis may be due to other effects, probably to recovery phenomena