Function and Regulation of Cargo Recognition by Clathrin Adaptors

真核细胞内膜封闭腔室的有序运动对正常功能至关重要。有许多不同种类的细胞膜腔室，每一个都有自己独特的生物发生和细胞内放置和运动的分子机制。这种隔室的一个子集利用一种分子机制来形成和运输，其中包括一种叫做网格蛋白的蛋白质，它在膜隔室周围形成一个短暂的“外衣”或“篮子”。这种被网格蛋白包裹的膜室参与了细胞外环境中特定物质的内化（受体介导的内吞作用）以及新合成的蛋白质向细胞表面的运动。因此，细胞内“网格蛋白包被囊泡”的种群是具有不同内容物的室室的异质混合物，因此必然有不同的目的地。这个项目关注的是理解细胞如何“知道”这样一个隔室的内容，以便它“知道”将其发送到哪里。网格蛋白衔接蛋白复合物AP2和AP1分别是质膜和反式高尔基网络（TGN）上的网格蛋白包被的主要成分，参与网格蛋白包被囊泡的形成。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招募被包裹的囊泡运输的整体膜蛋白（“货物”）。APs与货物的相互作用也涉及衣层的组装。尽管对AP功能的生物化学和细胞生物学特性已经有了相当多的研究，但它们在膜交通中的作用仍有许多方面尚未确定。特别是，ap -货物相互作用在细胞功能中的作用直到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APs如何通过结合受体细胞质结构域的序列基序（如YxxQ，其中x是任何氨基酸，Q是一个大体积的疏水残基）来识别货物。其他人已经定义了许多与细胞内ap相互作用的调节蛋白。从细胞中AP定位的研究可以清楚地看出，这些分子在其功能位点与细胞膜有非常特定的相互作用。然而，它们在细胞中不同部位发挥作用的能力有多少是由货物识别决定的，这一点尚未确定。Sorkin博士的实验室开发了一种独特的系统，用于检测蛋白质分选过程中AP2和AP1的需求。在这个系统中，他们成功地用相同蛋白的突变版本取代了AP复合物的内源性m1和m2亚基。具体来说，他们突变了AP2 m2亚基的货物识别位点，并产生了表达突变AP2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的表达消除了一些质膜受体的摄取，但不影响其他物质的内吞作用。这些数据强调了仔细评估ap -货物相互作用在体内作用的重要性。本项目将扩展这些研究，更深入地分析m2突变对网格蛋白包覆的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AP1功能中的作用。m1和m2在不同细胞位置的比较作用将通过两者之间嵌合分子的产生和分析表达这些嵌合的细胞中的膜交通来确定。第一个目标是验证假设，即与YxxQ基序的相互作用对货物分拣至关重要，对AP2的靶向和对接的影响可以忽略不计，但对AP1外壳的组装很重要。为此，将利用表达AP2或AP1突变体m2或m1亚基的细胞来评估m与含yxxq蛋白的相互作用在APs的正确靶向以及细胞表面和TGN的包被坑/芽组装中的作用。第二个目的是通过分析无法识别YxxQ基序的AP1突变m1亚基的表达影响，验证AP1在tgn -内体双向运输中发挥作用的假设。在目标3中，m1和m2在不同细胞位置的比较作用也将通过两者之间嵌合分子的产生和表达这些嵌合体的细胞中的膜交通分析来研究。这些研究将确定ap -货物相互作用在指导膜运输中的作用，并揭示沿内吞和分泌途径的蛋白质分选调节机制的新见解。

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.