Exol and Exo2 proteins stimulate calcium-dependent exocytosis in permeabilized adrenal chromaff in cells

Abstract

IN many cell types an increase in cytosolic calcium is the main signal for the exocytotic release of stored secretory components such as hormones and neurotransmitters. The site of action of calcium in exocytosis is not known, neither are the participating molecules^1,2. In the case of the intracellular membrane fusions that occur during transport through early stages of the secretory pathway, several cytosolic and peripheral membrane proteins are necessary^3–6. Permeabilized cells have been useful in understanding the requirements for calcium and nucleotides in regulated exocytosis^7,8 and under certain conditions there is leakage of soluble protein components and run-down of the exocytotic response^9–14. This system can be used to identify the soluble proteins involved in exocytosis, one candidate in chromaffin cells being annexin II (calpactin)⁹. Here we use this assay to identify two other cytosolic protein factors that regulate exocytosis in permeabilized adrenal chromaffin cells, which we term Exol and Exo2. Exol from brain cytosol resolves on electrophoresis in SDS–polyacrylamide gels as a group of polypeptides of relative molecular mass ˜30,000 and shares sequence homology with the 14–3–3 family of proteins. The ability of Exol to reactivate exocytosis is potentiated by protein kinase C activation and there-fore Exol may influence the protein kinase C-mediated control of Ca²⁺-dependent exocytosis.