The unicellular eukaryote Paramecium can be stimulated to release synchronously thousands of secretory vesicles. Parafusin (PFUS), a cytosolic phosphoglycoprotein, dephosphorylates and rephosphorylates in a calcium dependent manner during exocytosis. PFUS has been cloned and the cDNA deduced amino acid sequence showed a 50.7% identity to rabbit muscle phosphoglucomutase PFUS, however, has major differences such as 4 insertions and 2 deletions Its localization was studied in both wt and exo" mutant Paramecium with an affinity purified peptide antibody (RA1, against an insertion) using indirect immunotluorescence and confocal microscopy. In wt, PFUS localized to secretory vesicles as rows of rings ( 1 urn in diameter) below the cell membrane in cross-section and as hollow barrels ( 3 um in length) in longitudinal section. In addition, rows of fluorescent dots at the cell membrane and diffuse "cytosolic" stain were observed Double staining with RA1 and a monoclonal antibody against the secretory product showed that PFUS localized to the exterior and not the interior of the vesicle. Western blot analysis confirmed that PFUS was not associated with the released secretory product. In ts mutant nd9 grown at restrictive temperature (containing docked but non-releasing secretory vesicles) PFUS showed a similar staining patterns as in wt except the rows of dots at the cell membrane were less organized. In mutant tam8 (blocked transport of secretory vesicles ) no staining was observed near the cell surface region; however, a few undocked vesicles in the cytosol showed barrel staining. These results indicate that PFUS associates with secretory vesicles, probably prior to docking, and the cell membrane, thus suggesting a possible involvement of PFUS during exocytosis.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Molecular Biology