Parafusin as part of the fusion machinery in Paramecium

Abstract

This thesis is focused on elucidating the role of the cytosolic Phosphoglycoprotein, parafusin (PFUS), in the important cellular process of regulated exocytosis in Paramecium tetraurelia using wt and secretory (exo -) mutants nd6, nd9 and tam8.;Previous studies have shown that PFUS, a 63kD protein, dephosphorylates in a Ca2+-dependent manner during exocytosis and this dephosphorylation is likely to be a dephosphoglucosylation by a Ca 2+ activated phosphodiesterase. The enzyme or its activation is defective in the mutant nd9-27°C cells. The cDNA of PFUS shows homology to mammalian phosphoglucomutase (PGM) with major insertions, deletions and a different N-terminus. A mammalian homologue of PFUS has been found in beta-TC3 cells. These results suggest that PFUS dephosphoglucosylation is coupled to the signal transduction pathway leading to membrane fusion and exocytosis.;Genetic analysis showed that there were at least two copies of parafusin/PGM-related genes in Paramecium and they were expressed. In addition, four extremely small introns were found in the gene we studied.;PFUS was present in pellet cell fractions suggesting interaction with membrane(s). Localization was studied using laser confocal scanning microscopy. In wt cells, PFUS was associated both with the exocytic sites of the cell membrane as rows of dots and with the membrane of the dense core secretory vesicles as a "coat". Localization was confirmed by studies in the mutant tam8. Association of PFUS with the membrane of the vesicles was confirmed since: (1) in double labeling experiment, PFUS outlined the content staining; (2) isolated intact secretory vesicles but not the released contents showed PFUS association. Whole protein antibody staining revealed association with alveolar sac in addition. These results again suggested that there were multiple proteins (isoforms) localized to different cellular compartments. Upon stimulation of exocytosis, PFUS localization changed only to reappear with recovery of exocytosis. PFUS expression levels did not change during this process.;To further dissect the temporal sequence of PFUS dephosphorylation and redistribution, studies were carried out in the exo-mutants nd6 and tam8. Unlike the nd9-27°C cells, both mutants dephosphorylated upon stimulation but PFUS localizations did not change. Thus dephosphorylation probably preceded redistribution and exocytosis and therefore could be necessary but not sufficient for either process.;Para-nitrophenol (pNP) stimulates cells to pseudoexocytosis (vesicle matrix expands without vesicle and cell membrane fusion) independent of Ca 2+. After pNP stimulation PFUS did not dephosphorylate and remained associated with the cell membrane. Thus PFUS is not required for matrix expansion but is probably required for membrane fusion. Some of the released matrixes attached to area showed PFUS localization as dots, confirming PFUS association with exocytic sites.;PFUS associates with both fusion partner membranes. Upon stimulation, PFUS dephosphoglucosylates and dissociates from both membranes. We thus suggest that although PFUS is structurally related to PGM, it is a novel critical component whose cycling probably participates in the molecular exocytic fusion machinery in Paramecium.