Regulation of clathrin adaptor complexes

网格蛋白接头蛋白（AP）复合物通过识别特定细胞器上的货物，并招募外壳成分形成运输囊泡来调节蛋白质运输。我们的长期目标是确定调节AP复合物不同功能的机制。在本提案中，我们将描述调节AP-1复合物在介导高尔基体/内体运输中的活性的新蛋白质，重点关注促进外壳组装和膜曲率的脂质修饰酶。******脂质翻转酶Drs2产生的膜不对称在AP-1囊泡的形成中起关键作用，反过来，AP-1调节Drs2的活性。然而，这种相互调节的机制还没有得到很好的理解。此外，我们最近发现预测的磷脂酶Mil1是一种新的调节蛋白，它调节由亚基异构体的不同使用而产生的功能不同的AP-1R复合体。Mil1和Drs2可能共同调节不同类型AP-1囊泡的形成。然而，许多问题仍然存在。Drs2进入AP-1囊泡是如何调控其活性的？Drs2是否同时并入AP-1和AP-1R囊泡？Mil1的作用是什么，是否与Drs2协同产生膜曲率？在这里，我们将研究Mil1， Drs2和囊泡出芽机制元件之间的关系，以了解翻转酶和脂质重塑过程如何协同驱动膜曲率。******许多翻转酶具有被调节蛋白结合取代的自身抑制序列。我们将确定激活转运活性的构象变化是否需要AP-1结合。我们还将研究AP-1是否通过典型的载物结合口袋与Drs2结合，以及Drs2是否与AP-1和AP-1R表现出不同的相互作用。******我们还将研究Mil1的功能作用。我们将描述Mil1的催化活性和底物特异性，并确定该蛋白如何介导接头招募和/或随后的囊泡出芽事件。此外，我们将研究Mil1的人类同源物是否调节AP-1的功能，或在高等细胞的其他囊泡运输步骤中发挥作用。******这些实验将进一步确定AP-1与Drs2翻转酶之间的关系，并将确定脂质修饰蛋白在AP-1囊泡形成中的作用。最近的研究发现了其他与涂层相关的脂质重塑蛋白，这些蛋白可能会产生囊泡形成所需的膜曲率。因此，这项工作将为控制膜曲率和囊泡形成的保守过程提供新的见解，并可能增加我们对脂质重塑和脂质不对称如何共同指导细胞内运输的理解

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.**