Homotypic fusion of immature secretory granules during maturation in a cell-free assay

The biogenesis of secretory granules embodies several morphological and bio chemical changes. In particular, in neuroendocrine cells maturation of secr etory granules is characterized by an increase in size which has been propo sed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconsti tutes homotypic fusion between one population of ISGs containing a [S-35]su lfate-labeled substrate, secretogranin II (SgII), and a second population c ontaining the prohormone convertase PC2. Both substrate and enzyme are targ eted exclusively to ISGs. Fusion is measured by quantification of a cleavag e product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependen t, and requires ATP and GTP and cytosolic proteins, NSF (N-ethylmaleimide-s ensitive fusion protein) is amongst the cytosolic proteins required, wherea s we could not detect a requirement for p97. The ability to reconstitute IS G fusion in a cell-fee assay is an important advance towards the identifica tion of molecules involved in the maturation of secretory granules and will increase our understanding of this process.