Regulation of clathrin adaptor complexes

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Clathrin adaptor protein (AP) complexes regulate protein trafficking by recognizing cargo at specific organelles, and recruiting coat components to make a transport vesicle. Our long term objective is to identify mechanisms that regulate the differential functions of AP complexes. In this proposal, we will characterize novel proteins that regulate the activity of the AP-1 complex in mediating Golgi/endosomal transport, with a focus on lipid-modifying enzymes that promote coat assembly and membrane curvature.******Membrane asymmetry created by the lipid flippase Drs2 plays a key role in the formation of AP-1 vesicles, and conversely, AP-1 regulates the activity of Drs2. However, the mechanisms involved in this reciprocal regulation are not well understood. Moreover, we have recently identified the predicted phospholipase Mil1 as a novel regulatory protein that regulates a functionally distinct AP-1R complex generated by the differential use of subunit isoforms. Mil1 and Drs2 may work together to regulate the formation of different classes of AP-1 vesicles. However, many questions remain. How does the sorting of Drs2 into AP-1 vesicles regulate its activity? Is Drs2 incorporated into both AP-1 and AP-1R vesicles? What is the function of Mil1, and does it cooperate with Drs2 to generate membrane curvature? Here, we will examine the relationship between Mil1, Drs2 and elements of the vesicle budding machinery to understand how flippases and lipid remodelling processes cooperate to drive membrane curvature. ******Many flippases have autoinhibitory sequences that are displaced by the binding of regulatory proteins. We will determine if the conformational change that activates transport activity is required for AP-1 binding. We will also investigate if AP-1 binds Drs2 via a canonical cargo-binding pocket, and if Drs2 displays differential interactions with AP-1 and AP-1R.******We will also investigate the functional role of Mil1. We will characterize Mil1's catalytic activity and substrate specificity, and determine how this protein mediates adaptor recruitment and/or subsequent vesicle budding events. Moreover, we will investigate if the human homolog of Mil1 regulates AP-1 function, or plays a role in other vesicle transport steps in higher cells.******These experiments will further define the relationship between AP-1 and the Drs2 flippase, and will determine the role of lipid- modifying proteins in AP-1 vesicle formation. Recent studies have discovered other coat-associated lipid remodeling proteins that may create the membrane curvature needed for vesicle formation. Thus, this work will give new insights into conserved processes that control membrane curvature and vesicle formation and may add to our understanding of how lipid remodeling and lipid asymmetry work together to direct intracellular transport.**