Combined targeted Omic and Functional Assays Identify Phospholipases A₂ that Regulate Docking/Priming in...

The fundamental molecular mechanism underlying the membrane merger steps of regulated exocytosis is highly conserved across cell types. Although involvement of Phospholipase A₂ (PLA₂) 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, PLA₂ were found to be associated with release-ready cortical secretory vesicles (CV) that possess the minimal molecular machinery for docking, Ca²⁺ 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 PLA₂ and a luminal secretory PLA₂ significantly reduce docking/priming in the late steps of regulated exocytosis, indicating key regulatory roles in the critical step(s) preceding membrane merger.