WT and mutant analysis of the ca2+-dependent dephosphorylation and dissociation of parafusin upon exocytosis

In the unicellular eukaryote Paramecium parafusin (PFUS) has been shown to dephosphorylate and dissociate from the exocytic site and the dene core secretory vesicles in a Oa2+-dependent manner upon exocytosis (Zhao and Salir. Kur. J. C'ell biol., 1998). During recovery of exocytosis PFUS re-associates with the vesicles in the cytoplasm prior to docking. The temporal sequence of these two events have been investigated using a series of axenic culture of exo " mutants (tamH. defective in vesicle transport; nd6, vesicles docked blocked in exocytosis; and nd9, ts mutant) combined with m vivo phosphoryiaikm experiments (incorporation of 32P,) and immunolocalization of PFl'S (using a PFUS specific peptide antibody. 1-2) in laser scanning con focal microscopy. In all in vivo phosphorylation experiments stimulation was performed with trinitrophenol following the original protocol whereas in the immunolocalization studies the more physiological stimulus, lysozyme. was used. Both give the .same results but cell morphology is much better preserved with lysozyme. Stimulation of nd9 cells with extracellular Ca2+ as expected showed no exocytosis nor tlephosphorylation and no change in PFUS association with the cell ;uid vesicle membranes. However, when tamS and ndö relis were stimulated with extracellular Ca2+ the following results were obtained: neither mutant <>Nor\iosed nor lost the PFUS association with the two membranes but. in both mutant:-, PFUS dephosphorylated. These results suggest that PFUS dephosplmrybiîion precedes exocytosis and may be necessary but not sufficient for PFUS M'dUtribution.