Function and Regulation of Cargo Recognition by Clathrin Adaptors

真核细胞内膜封闭区室的有序运动对正常功能至关重要。 存在许多不同种类的这样的细胞内膜隔室，每种都具有其自身独特的生物发生和细胞内放置和移动的分子机制。 这些隔室的一个子集利用分子机制来形成和运输，其涉及称为网格蛋白的蛋白质，其在膜隔室周围形成短暂的“涂层”或“篮”。 这种网格蛋白包被的膜隔室参与来自细胞外环境的特定物质的内化（受体介导的内吞作用）和新合成的蛋白质向细胞表面的移动。 因此，细胞内的“网格蛋白包被的囊泡”群体是具有不同内容物的隔室的异质混合物，因此必然具有不同的目的地。 该项目关注的是了解细胞如何“知道”这样一个隔间的内容，使它“知道”在哪里发送it. Clatherin适配器蛋白复合物AP 2和AP 1的主要组成部分，网格蛋白涂层在质膜和trans-Golgi网络（TGN），分别参与形成网格蛋白涂层囊泡。 这些囊泡负责受体介导的内吞作用和细胞器生物发生的基本细胞功能。 AP复合物的关键功能之一是选择性地募集由包被囊泡转运的整合膜蛋白（“货物”）。 AP与货物的相互作用也涉及涂层的组装。 尽管AP功能的生物化学和细胞生物学的表征相当多，但它们在膜交通中的作用的许多方面尚未被定义。 特别是，AP-货物相互作用在其细胞功能中的作用最近才开始通过初步实验来解决，这些实验构成了该项目的基础。 几个实验室已经通过体外实验确定了AP如何通过结合受体胞质结构域中的序列基序（如YxxQ，其中x是任何氨基酸，Q是大的疏水残基）来识别货物。其他人已经定义了许多与细胞中的AP相互作用的调节蛋白。 从细胞中AP定位的研究中可以清楚地看出，这些分子在其功能位点与细胞内膜具有非常特异的相互作用。 然而，还没有确定它们在细胞中不同位点发挥功能的能力有多少是由货物识别决定的。 Sorkin博士的实验室开发了一种独特的系统，用于检查蛋白质分选中对AP 2和AP 1的要求。在这个系统中，他们已经成功地用相同蛋白质的突变形式取代了AP复合物的内源性m1和m2亚基。 具体来说，他们突变了AP 2的m2亚基的货物识别位点，并产生了表达突变的AP 2复合物的细胞，这使他们能够研究AP-货物相互作用在决定AP定位和功能中的作用。 突变体m2的表达取消了一些质膜受体的摄取，但不影响其他货物的内吞。 这些数据突出了仔细评估AP-货物相互作用在体内的作用的重要性。该项目将扩展这些研究，更深入地分析m2突变对网格蛋白包被的小坑形成和货物靶向的影响，并应用相同的策略来阐明m1亚基在AP 1功能中的作用。M1和M2在不同细胞位置的比较作用将通过产生两者之间的嵌合分子并分析表达这些嵌合体的细胞中的膜交通来定义。第一个目的是检验与YxxQ基序的相互作用对货物分选至关重要，对AP 2的靶向和对接的影响可以忽略不计，但对AP 1外壳的组装很重要的假设。为此，相应地，将利用表达AP 2或AP 1的突变m2或m1亚基的细胞来评估m与含YxxQ的蛋白质的相互作用在AP的正确靶向以及在细胞表面和TGN处的包被的凹坑/芽的组装中的作用。第二个目的是通过分析不能识别YxxQ基序的AP 1突变体m1亚基表达的影响来检验AP 1在双向TGN-内体运输中起作用的假设。在目标3中，还将通过产生两者之间的嵌合分子并分析表达这些嵌合体的细胞中的膜交通来研究m1和m2在不同细胞位置中的比较作用。这些研究将确立AP-货物相互作用在引导膜运输中的作用，并揭示蛋白质分选沿着内吞和分泌途径的调节机制的新见解。

The orderly movement of membrane-enclosed compartments within the eukaryotic cell is critical to proper function. There are many different kinds of such intracellular membrane compartments, each with its own unique molecular mechanisms for biogenesis and intracellular placement and movement. A subset of such compartments utilizes a molecular mechanism for formation and trafficking that involves a protein called clathrin which forms a transient "coat" or "basket" around the membrane compartment. Such clathrin-coated membrane compartments are involved in the internalization of specific materials from the extracellular milieu (receptor-mediated endocytosis) and in the movement of newly synthesized proteins out to the cell surface. Thus, the population of "clathrin-coated vesicles" inside the cell is a heterogeneous mixture of compartments with different contents and therefore necessarily different destinations. This project concerns itself with understanding how the cell "knows" the content of such a compartment so that it "knows" where to send it. Clathrin adaptor protein complexes AP2 and AP1 are major components of clathrin coats at the plasma membrane and trans-Golgi network (TGN), respectively, where they participate in formation of clathrin-coated vesicles. These vesicles are responsible for the basic cellular functions of receptor-mediated endocytosis and organelle biogenesis. One of the key functions of AP complexes is to selectively recruit the integral membrane proteins ('cargo') transported by coated vesicles. The interaction of APs with cargoes is also implicated in the assembly of coats. In spite of a considerable amount of characterization of the biochemistry and cell biology of AP function, there are many aspects of their roles in membrane traffic that have yet to be defined. In particular, the role of AP-cargo interactions in their cellular function has only recently begun to be addressed through preliminary experiments that form the basis for this project. Several laboratories have established through in vitro experiments how APs recognize cargo by binding to sequence motifs (such as YxxQ, where x is any amino acid and Q is a bulky hydrophobic residue) in the cytoplasmic domains of receptors. Others have defined numerous regulatory proteins which interact with APs in cells. It is clear from studies of AP localization in cells that these molecules have very specific interactions with intracellular membranes at the sites of their function. It is not established, however, how much of their ability to function at different sites in the cell is determined by cargo recognition. Dr. Sorkin's lab has developed a unique system for examining the requirements for both AP2 and AP1 in protein sorting. In this system, they have successfully replaced the endogenous m1 and m2 subunits of the AP complexes by mutated versions of the same proteins. Specifically, they have mutated the cargo recognition site of the m2 subunit of AP2 and produced cells expressing the mutant AP2 complex, which allowed them to investigate the role of AP-cargo interactions in dictating AP localization and function. The expression of mutant m2 abolished the uptake of some plasma membrane receptors but do not affect the endocytosis of other cargoes. These data highlight the importance of a careful evaluation of the role of AP-cargo interaction in vivo. This project will extend these studies to a more in depth analysis of the effects of the m2 mutation on clathrin-coated pit formation and cargo targeting, and apply the same strategy to elucidate the role of m1 subunit in AP1 function. The comparative roles of m1 and m2 in different cellular locations will be defined by production of chimeric molecules between the two and analyzing membrane traffic in cells expressing these chimeras. The first objective is to test the hypothesis that interactions with YxxQ motifs, that are critical for cargo sorting, have negligible influence on targeting and docking of AP2 but is important for the assembly of AP1 coats. To this end, cells expressing mutant m2 or m1 subunit of AP2 or AP1, correspondingly, will be utilized to assess the role of m interactions with YxxQ-containing proteins in the correct targeting of APs and the assembly of coated pits/buds at the cell surface and TGN. The second objective is to test the hypothesis that AP1 plays a role in bi-directional TGN-endosomal trafficking through analysis of the effects of expression of mutant m1 subunits of AP1 incapable of YxxQ motif recognition. The comparative roles of m1 and m2 in different cellular locations will also be investigated in Objective 3 by production of chimeric molecules between the two and analyzing membrane traffic in cells expressing these chimeras. These studies will establish the role of AP-cargo interactions in directing membrane traffic and uncover new insights into regulatory mechanisms of protein sorting along the endocytic and secretory pathways.