Synaptotagmin IV function in secretory granule maturation in neuroendocrine cells
In neuroendocrine cells, immature secretory granules (ISGs) bud from the trans Golgi network (TGN), and then undergo a maturation process that includes ISG-ISG homotypic fusion, removal of excess membranes via clathrin-coated vesicles (CCVs) and acidification of the granules. These mature secretory granules (MSGs) can then fuse with the plasma membrane upon receiving an external signal, and finally release their content in the extracellular space. In this thesis, I will present evidence that Synaptotagmin IV (Syt IV) is one of the components involved in the regulation of granule maturation in neuroendocrine cells. Syt IV is a neuron and neuroendocrine cell-specific isoform, which belongs to the synaptotagmin family of membrane trafficking proteins. After confirming that Syt IV is localised on ISGs and absent from MSGs in PCI2 cells, I investigated whether Syt IV is involved in ISG homotypic fusion by adding the purified Syt IV cytoplasmic domain into an in vitro assay, where it functions as a dominant negative. Addition of this domain, but not a similar domain from Syt I, resulted in a dose-dependent inhibition of ISG-ISG fusion. Furthermore, I found that Syt IV binds to the ISG-SNARE Syntaxin 6 (Stx6), suggesting that the two proteins might be part of the same machinery that regulates ISG maturation. In addition, I used an in vivo approach based on the processing of secretogranin II (Sgll) into its degradation product pi8, which occurs during ISG maturation, to assay Syt IV function. I show that the Syt IV cytoplasmic domain, as well as siRNA-mediated knockdown of Syt IV inhibits Sgll processing by prohormone convertase 2 (PC2). Interestingly, PC2 is found mostly in the pro-form, suggesting that activation of PC2 is also inhibited. We conclude that Syt IV is an essential component for the process of secretory granule maturation. Lastly, I found that Syt IV binds preferentially to membrane-bound clathrin adaptor protein-1 (AP-1). This suggests that Syt IV might be sorted from maturing SGs in a complex, possibly via an ISG-SNARE protein that interacts directly with AP-1.