Regulation of clathrin adaptor complexes

网格蛋白衔接蛋白（AP）复合物通过识别特定细胞器上的货物，并募集外壳成分以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复合物的差异功能的机制。在这项提案中，我们将描述新的蛋白质，调节AP-1复合物的活性，介导高尔基体/内体转运，重点是脂质修饰酶，促进外套组装和膜曲率。 由脂质翻转酶Drs 2产生的膜不对称性在AP β 1囊泡的形成中起着关键作用，相反，AP β 1调节Drs 2的活性。然而，这种相互调节的机制还不清楚。此外，我们最近已经确定了预测的磷脂酶Mil 1作为一种新的调节蛋白，调节功能不同的AP-1 R复合物产生的亚基异构体的差异使用。Mil 1和Drs 2可能共同调节不同类型AP β 1囊泡的形成。然而，许多问题仍然存在。Drs 2如何被分选到AP β 1囊泡中调节其活性？Drs 2是否同时整合到AP β 1和AP β 1 R囊泡中？Mil 1的功能是什么，它是否与Drs 2合作产生膜曲率？在这里，我们将研究Mil 1，Drs 2和囊泡出芽机制的元素之间的关系，以了解翻转酶和脂质重塑过程如何合作，以驱动膜曲率。 许多翻转酶具有通过调节蛋白的结合而被置换的自抑制序列。我们将确定激活转运活性的构象变化是否是AP-1结合所必需的。我们还将研究AP β 1是否通过典型的货物β 1结合口袋结合Drs 2，以及Drs 2是否与AP β 1和AP β 1 R显示差异相互作用。 我们还将研究Mil 1的功能作用。我们将描述Mil 1的催化活性和底物特异性，并确定这种蛋白质如何介导适配器招聘和/或随后的囊泡出芽事件。此外，我们将研究是否人类同源的Mil 1调节AP-1的功能，或在其他囊泡运输步骤中发挥作用，在高等细胞。 这些实验将进一步确定AP β 1和Drs 2翻转酶之间的关系，并将确定脂质修饰蛋白在AP β 1囊泡形成中的作用。最近的研究发现了其他与囊泡相关的脂质重塑蛋白，它们可能会产生囊泡形成所需的膜曲率。因此，这项工作将提供新的见解保守的过程，控制膜曲率和囊泡的形成，并可能增加我们的理解如何脂质重塑和脂质不对称性一起工作，直接细胞内运输。

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 AP­1 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 AP­1 vesicles, and conversely, AP­1 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 AP­1R complex generated by the differential use of subunit isoforms. Mil1 and Drs2 may work together to regulate the formation of different classes of AP­1 vesicles. However, many questions remain. How does the sorting of Drs2 into AP­1 vesicles regulate its activity? Is Drs2 incorporated into both AP­1 and AP­1R 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 AP­1 binds Drs2 via a canonical cargo­binding pocket, and if Drs2 displays differential interactions with AP­1 and AP­1R. 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 AP­1 function, or plays a role in other vesicle transport steps in higher cells. These experiments will further define the relationship between AP­1 and the Drs2 flippase, and will determine the role of lipid­ modifying proteins in AP­1 vesicle formation. Recent studies have discovered other coat­associated 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.