Parafusin is a membrane and vesicle associated protein that cycles at exocytosis.
In the unicellular eukaryote Paramecium tetraurelia, stimulation of exocytosis leads to Ca2+ activation of an alpha Glc-1-phosphodiesterase that dephosphoglucosylates the phosphoglycoprotein parafusin (PFUS). This process fails in exo mutant nd9 and also in the absence of Ca2+ influx upon stimulation suggesting that PFUS dephosphoglucosylation may be causally related to exocytosis. To further corroborate the hypothesis that PFUS is involved in the molecular events in exocytosis, we used laser confocal scanning microscopy and a PFUS specific peptide antibody to perform localization studies of PFUS in wild type (wt) and mutant Paramecium. In unstimulated wt cells, PFUS was associated both with the exocytic site of the cell membrane and with the membrane of the dense core secretory vesicles. Localization at these two sites was shown to be independent of each other since the exocytosis mutant (exo-) tam8, in which docking of its vesicles is blocked, still showed cell membrane staining. Immunofluorescence and immunoblotting of isolated intact secretory vesicles also revealed PFUS association. Upon stimulation of exocytosis, PFUS dissociated from both the dense core secretory vesicles and the cell membrane in a Ca(2+)-dependent manner. During recovery of exocytic capacity, PFUS reassociated with the newly formed secretory vesicles in the cytoplasm prior to their docking at the exocytic sites. Immunoblot analysis of PFUS during this time showed no changes in levels of the protein. Stimulation of exocytosis in wt in Mg2+ buffer or in the exo- temperature sensitive mutant (nd9) at the non-permissive temperature did not lead to dissociation of the PFUS. We conclude that PFUS is a novel critical component whose cycling probably participates in the molecular exocytic fusion machinery in these cells.
Zhao
H
H
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY 10461, USA.
Satir
B H
BH
D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D005089:Exocytosis / Q000235:genetics / Q000502:physiology*; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D017830:Octoxynol; D016805:Paramecium tetraurelia / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010733:Phosphoglucomutase; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D010750:Phosphoproteins; D015800:Protozoan Proteins; C087273:parafusin protein, Paramecium; D017830:Octoxynol; D010733:Phosphoglucomutase