RNA interference of PFUS results in inhibition of secretory vesicle biogenesis

Abstract

In protistan evolution, gene duplication gives rise to phosphoglucomutase (PGM) superfamily members which had virtually no PGM activity, including Paramecium parafusin (PFUS) and its Toxoplasma and mammalian orthologs, phosphoglycoproteins whose role in Ca2+ ‐regulated exocytosis has been controversial. We show that RNAi knockdown of PFUS leads to an exo− phenotype. Three different regions of the PFUS sequence were targeted. As a control we used the empty RNAi vector. Exocytosis was scored after stimulation. Overall, exocytosis becomes inhibited with all three RNAi constructs, compared to the control. In order to examine if inhibition correlated with a reduction of PFUS, we performed western blot analysis using a polyclonal antibody which recognizes all Paramecium proteins in the PGM superfamily (pan‐PGM antibody) and a synthetic peptide antibody, PFUS antibody, which recognizes PFUS specifically. Cells exposed to RNAi for 72 hr show that PFUS decreases, whereas no decrease is seen in other PGM isoforms. Double labeling in confocal microscopy for PFUS and secretory content (T‐8) shows a corresponding decrease in dense core secretory vesicles (DCSVs) in the cells. These changes are reversed by re‐feeding in the absence of PFUS RNAi. These data demonstrate that knockdown of PFUS inhibits exocytosis, probably by blocking DCSV packaging.