STUDIES ON EXOCYTOSIS IN PARAMECIUM TETRAURELIA: ROLE OF DIVALENT CATIONS AND PROTEIN PHOSPHORYLATION (PHOSPHOPROTEIN, CALCIUM-DEPENDENT, PROTOZOA, TRICHOCYST, STIMULUS-SECRETION COUPLING)

Abstract

The cortex of Paramecium contains thousands of membrane-bound secretory organelles called trichocysts, whose content, the trichocyst matrix (tmx), is released in response to secretagogues (e.g. picric acid, paranitrophenol or Alcian Blue). Studies with light microscopy, freeze fracture (FFEM) and transmission (TEM) electron microscopy show that high extracellular Mg('2+) inhibits two morphologically characterized secretory events, membrane fusion (exocytosis) and tmx expansion, suggesting that Ca('2+) is required for both.;Exocytotic sites in the plasma membrane are characterized in FFEM with specific intramembrane particle arrays (rosettes). A temperature sensitive secretory mutant, nd 9, lacks rosettes and does not secrete when grown at 27(DEGREES)C, but assembles rosettes and does secrete when grown at 18(DEGREES)C. The divalent cation ionophore A23187, which bypasses physiologically regulated Ca('2+) influx, triggers normal secretion in wild type (wt) cells, but only tmx expansion (without membrane fusion) in non-permissive mutants, suggesting that the rosette particles represent membrane components specifically required for exocytosis.;Exocytosis is correlated with the Ca('2+)-dependent dephosphorylation of a 65,000 M(,r) polypeptide (65K) in two types of experiment: (1) high extracellular Mg('2+) inhibits picric acid-induced secretion and dephosphorylation of 65K in wt cells, and (2) nd 9 permissive cells secrete and dephosphorylate 65K in response to picric acid, whereas nd 9 non-permissive cells do not. Thus the presence of an assembled rosette is required for the Ca('2+)-dependent dephosphorylation of 65K.;The effects of paranitrophenol and Alcian Blue on membrane fusion, tmx expansion and dephosphorylation of 65K in wt and nd 9 mutants are also examined. Further information about the stimulus-sensitive phosphoprotein is obtained from subcellular fractionation, in vitro phosphorylation, two dimensional gel electrophoresis, and phosphoamino acid identification. Results suggest that the 65K phosphoprotein may be a component of the molecular pathway coordinating exocytosis in response to extracellular stimulation.