Parafusin, a super-family member of phosphoglucomutase, is crucial for the function of the calcium-dependent exocytic nano-machinery
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Parafusin (PFUS), a superfamily member of phosphoglucomutase (PGM), is involved in the exocytic nano-machinery. It is a 63kD phosphoglycoprotein with two cyclic covalent modifications (phosphorylation/dephosphorylation and phosphoglucosylation/dephosphoglucosylation). It is a component of the scaffold associated with dense core secretory vesicles (DCSV). This thesis focuses on the role of PFUS and its post-translational modifications in exocytosis.;To conclusively establish the importance of PFUS in exocytosis, PFUS RNAi was performed in Paramecium by bacteria feeding. Cells exposed to RNAi showed loss of exocytosis (60-70%) after 3-4 generations of growth (72 hr). Using pan-PGM and PFUS antibodies on western blots, PFUS was shown to be specifically knocked-down without obvious change in overall PFUS/PGM level. Immunofluorescent localization in RNAi cells showed reduction of PFUS synthesis and number of DCSVs, indicating PFUS affects the secretory pathway. These cells recover exocytosis capacity after transfer to fresh medium. Inhibition of PFUS leads to an exo-phenotype, demonstrating that PFUS is an important component of the exocytic nano-machinery.;In order to examine the serine residue in PFUS post-translational modifications an ortholog assay in living Paramecium was developed. Molecular genetic methods are limited in Paramecium and it has different codon usage. Therefore, PRP1, PFUS ortholog in Toxoplasma was used. Electroporation of fluorescently labeled recombinant His-PRP 1 into live wt and tam8 (defective in vesicle transport) Paramecium showed that PRP1 localizes the same as endogenous PFUS. Upon exocytosis in wt Paramecium the electroporated His-PRP1 dissociates from DCSVs and re-associates as new vesicles are synthesized. These results correlate with those of endogenous PFUS.;In order to examine which specific residue(s) is involved, selected serines in His-PRP1 (S146, S243 and S560) was mutagenized and examined by in vitro phosphorylation. His-PRP1 (S146A or S146E) cannot be Ca 2+ ATP phosphorylated. In contrast, S243A and S560A both become phosphorylated. This indicates S146 is phosphorylated. When S146A and S146E are electroporated into living wt Paramecium, they remain cytoplasmic and do not associate with DCSVs. The positive control, S560A, with intact phosphorylation, still localizes to DCSVs. Thus serine S146 is an important residue for PRP1/PFUS vesicle association and function in the exocytic nano-machinery.
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