Homotypic fusion of immature secretory granules during maturation requiressyntaxin 6

Homotypic fusion of immature secretory granules (ISGs) gives rise to mature secretory granules (MSGs), the storage compartment in endocrine and neuroe ndocrine cells for hormones and neuropeptides. With the use of a cell-free fusion assay, we investigated which soluble N-ethylmaleimide-sensitive fusi on protein attachment receptor (SNARE) molecules are involved in the homoty pic fusion of ISGs. Interestingly, the SNARE molecules mediating the exocyt osis of MSGs in neuroendocrine cells, syntaxin 1, SNAP-25, and VAMP2, were not involved in homotypic ISG fusion. Instead, we have identified syntaxin 6 as a component of the core machinery responsible for homotypic ISG fusion . Subcellular fractionation studies and indirect immunofluorescence microsc opy show that syntaxin 6 is sorted away during the maturation of ISGs to MS Gs. Although, syntaxin 6 on ISG membranes is associated with SNAP-25 and SN AP-29/GS32, we could not find evidence that these target (t)-SNARE molecule s are involved in homotypic ISG fusion. Nor could we find any involvement f or the vesicle (v)-SNARE VAMP4, which is known to be associated with syntax in 6. Importantly, we have shown that homotypic fusion requires the functio n of syntaxin 6 on both donor as well as acceptor membranes, which suggests that t-t-SNARE interactions, either direct or indirect, may be required du ring fusion of ISG membranes.