Biogenesis and targeting of vesicles involved in regulated secretion of hormones and bioactive molecules are
important processes which are not well understood. Immature secretory granules (ISGs) are formed from the TGN
and mature during storage to mature secretory granules(MSGs). MSGs are the long-term storage compartment in
the cell which fuse in a regulated fashion with the plasma membrane to release the granule content.
The aim of this project is to gain information regarding one aspect maturation: homotypic fusion of ISGs.
Homotypic fusion occurs during maturation with the net result that ISGs increase in size. During maturation sorting
of the secretory granule membrane and colten occurs: This sorting is an active process which requires the
membrane remodelling. The hypothesis is that homotypic fusion and membrane remodelling are linked and result
in the formation of a MSG. Homotypic fusion of ISGs has been reconstituted in a cell-free assay (Urbé et al.,
submitted) and provides the ideal tool to study the molecules involved in fusion. Urbé et al. have shown that ISG-
ISG fusion occurs but not ISG-MSG fusion and that this process requires cytosol, NSF and ATP.
NSF activity requires soluble attachment proteins (SNAPs) and receptors (SNAREs). SNAREs are thought to
provide the specificity required for intracellular membranes to recognize each other. The questions to be addressed
are:
Which SNARs are responsible for the ISG-ISG homotypic fusion reaction? and how does the fusion machinery for
ISG homotypic fusion compare to that involved in fusion of MSGs with plasma membrane? To address these
questions I will use the cell-free assay first test known candidate SNAREs which are present on MSGs or the Golgi
complex for activity, and then if none are involved, search for novel SNAREs on ISGs.