Combined Targeted Omic and Functional Assays Identify Phospholipases A(2) that Regulate Docking/Priming in Calcium-Triggered Exocytosis

The fundamental molecular mechanism underlying the membrane merger steps of regulated exocytosis is highly conserved across cell types. Although involvement of Phospholipase A(2) (PLA(2)) in regulated exocytosis has long been suggested, its function or that of its metabolites—a lyso-phospholipid and a free fatty acid—remain somewhat speculative. Here, using a combined bioinformatics and top-down discovery proteomics approach, coupled with lipidomic analyses, PLA(2) were found to be associated with release-ready cortical secretory vesicles (CV) that possess the minimal molecular machinery for docking, Ca(2+) sensing and membrane fusion. Tightly coupling the molecular analyses with well-established quantitative fusion assays, we show for the first time that inhibition of a CV surface calcium independent intracellular PLA(2) and a luminal secretory PLA(2) significantly reduce docking/priming in the late steps of regulated exocytosis, indicating key regulatory roles in the critical step(s) preceding membrane merger.