Communication between cells in the nervous system depends on the regulated release of signalling molecules. In contrast to synaptic vesicles which store classical transmitters, the large dense core vesicle (LDCV), a component of the regulated secretory pathway stores neural peptides and hormones. Monoamine transmitters are also stored in LDCVs, further indicating the importance of this secretory pathway for major mental illness and drug abuse. The long term objective of this proposal is to understand how the storage of these neuromodulators contributes to information processing and psychiatric disease. The strategy is to use a transporter that packages monoamines into vesicles to understand the biogenesis of LDCVs and the regulated secretory pathway. Proteins destined for the regulated pathway sort away from constitutively released proteins at the level of the trans-Golgi network (TGN) but the mechanism remains unknown. Presumably, sorting involves the specific interaction of integral membrane proteins destined for the LDCV with a cytosolic sorting machinery. The laboratory has recently shown that a vesicular amine transporter (VMAT2) preferentially localizes to LDCVs rather than constitutive secretory vesicles or synaptic-like microvesicles acid so provides the first opportunity to study this important sorting event directly. In the first specific aim, we will use deletions and then point mutations to identify signals on VMAT2 required for sorting to LDCVs. To complement this approach and determine whether these sequences are sufficient for targeting to LDCVs, the second specific aim examines chimeras with another closely related transport protein that does not sort to LDCVs. The results of this work will provide some of the first information about the mechanism of sorting into the regulated secretory pathway and has important implications for the release of hormones, neural peptides and growth factors as well as monoamine transmitters.