? A central question to the biology of any cell, is how membrane proteins are specifically sorted by vesicle carriers. This problem is particularly pertinent to the synapse, a neuronal domain specialized in the trafficking of synaptic vesicles (SV). However, the mechansisms by which synaptic vesicle proteins make their sorting decisions are not well understood. To study this fundamental problem I have developed an adaptor-dependent neuronal vesiculation model. This mechanism is capable to sort from endosomes SV proteins using the adaptor complex AP-3 and two enzymes, a GTPase and an AP-3-associated kinase that phosphorylates the adaptor AP-3. Although phosphorylation has been recognized as an important cell regulatory mechanism, its role in adaptor function is mostly unexplored. The central hypothesis guiding this proposal is that an AP-3-associated kinase selectively regulates AP-3-dependent vesiculation and sorting functions. Our goal is to use unique cell-free reconstitution assays plus the analysis of synaptic vesicle protein trafficking in intact neurons to understand the basic mechanisms that govern SV protein sorting and to test the role of phosphorylation in controlling adaptor-dependent vesicle formation. These studies will reveal the first detailed model for the role of phosphorylation in an adaptor-dependent vesiculation process. In this proposal, we will convey the wealth of molecules, mechanisms and drugs that we have characterized in PC12 cells to dissect a novel endosome SV biogenetic pathway in neurons. In mice ablation of the AP-3 pathway results in pigment dilution, epilepsy, behavioral phenotypes and defective SV protein sorting whereas in humans generates the Hermansky-Pudlak II syndrome. The understanding of the AP-3 mechanisms and regulation will open avenues for the development of pathway-specific pharmacological agents that will impact the treatment of epilepsy, behavioral disorders and the Hermansky-Pudlak II syndrome. ? ?
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