Protein-protein interactions involving Munc18 in regulated exocytosis

Neurons, endocrine and many other cell types communicate with one another and other tissues in a co-ordinated manner by the tightly regulated release of transmitters and signalling molecules. Transmitters are packaged into small lipid vesicles which have to fuse with the cell membrane to release their contents only at the appropriate time and place. Most of the individual parts of the machinery inside the cell that allows this to occur have been discovered over the past ten years but we still have a very limited understanding of how the process works and how it is regulated. Changes in the functioning of the machinery for release are believed to contribute to learning and memory and affect behaviour. We aim to investigate in detail one essential component of the machinery, Munc18-1, by making specific modifications to its structure. This will allow us to learn how its interactions with other components contribute to its function in the various steps leading to transmitter release.

Release of neurotransmitters occurs by regulated exocytosis, whereby a rise in cytosolic Ca2+ triggers the fusion of a vesicle with the plasma membrane and the release of its content. Despite the fact that we know the identity of most of the proteins involved we have only a rudimentary view of the mechanisms or how the machinery is regulated. An increased knowledge of the exocytotic machinery will allow a full understanding of neurotransmitter release, synaptic plasticity and changes in neurotransmission in disease states. We will study Munc18-1, a protein essential for neurotransmitter release, a target for regulation of exocytosis by protein phosphorylation in which single amino acid changes lead to alterations in neurotransmission and behaviour. Models of Munc18-1 function are based in its high affinity interactions with the SNARE protein syntaxin. We will extend previous work characterising mutations in Munc18-1 by analysis of the effect on protein interactions in vitro and on exocytosis. We know that certain mutations that do not affect the interaction of Munc18-1 with its known binding partners, particularly syntaxin, do have marked effects on exocytosis. Further new mutants, including one with an impairment in Mint1 but not syntaxin binding, will be functionally characterised in protein interaction and exocytosis assays. We have identified Rab3A as a potential novel interacting partner for Munc18-1 and we will search for additional interacting partners using a variety of approaches. We aim to validate the Rab3A and any other interactions found and to characterise these interactions by a combination of in vitro, cellular and functional analyses that will allow us to assess the physiological signifiance of the protein-protein interactions.