Regulation of clathrin adaptor complexes

网格蛋白接头复合物的调节

• 批准号: RGPIN-2016-04290
• 负责人: Conibear, Elizabeth
• 金额: $ 3.21万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=614965
• 关键词: Regulation; clathrin; adaptor; complexes

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.

笼状蛋白适配器蛋白(AP)复合体通过识别特定细胞器上的货物，并招募外壳成分来形成运输囊泡来调节蛋白质的运输。我们的长期目标是确定调节AP复合体不同功能的机制。在这项提案中，我们将表征调节APü1复合体在介导高尔基体/内体转运中的活性的新蛋白质，重点是促进被膜组装和膜曲率的脂质修饰酶。 由脂翻转酶Drs2产生的膜不对称性在AP1囊泡的形成中起着关键作用，反过来，AP1调节DRS2的活性。然而，这种互惠监管涉及的机制还没有被很好地理解。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AP1R复合体。Mil1和Drs2可能共同作用来调节不同类别AP-1囊泡的形成。然而，许多问题仍然存在。DRS2在AP-1囊泡中的分类是如何调节其活性的？AP1和AP1R囊泡中都含有DRS2吗？Mil1的功能是什么，它是否与Drs2协同产生膜曲率？在这里，我们将研究Mil1，Drs2和囊泡出芽机制的元件之间的关系，以了解翻转酶和脂类重塑过程如何合作来驱动膜曲率。 许多Flipase具有被调节蛋白结合而移位的自身抑制序列。我们将确定激活转运活性的构象变化是否是AP-1结合所必需的。我们还将调查AP1是否通过规范的货物结合口袋与DRS2结合，以及DRS2是否与AP1和AP1R显示不同的相互作用。 我们还将研究Mil1的功能作用。我们将表征Mil1的S催化活性和底物特异性，并确定该蛋白如何介导适配器招募和/或随后的囊泡萌发事件。此外，我们将调查人类Mil1的同源物是否调节APü1的功能，或在高等细胞中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和Drs2 Flippase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，它们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作用以指导细胞内运输的理解。