Molecular Recognition by Clathrin Adaptors

笼状蛋白适配器的分子识别作用 在真核细胞的内吞作用和胞体分选中，网状蛋白被层起着普遍而基本的作用。网状蛋白形成一个笼子，包裹着载货的小泡，但网状蛋白本身并不直接与货物结合。货物通过连接货物和笼蛋白的接头蛋白被分类到覆盖着笼状蛋白的小泡中。最著名的通用接头是异四聚体接头蛋白复合体(AP复合体)和多模块GGA接头蛋白。该项目的总体目标是1)确定接头蛋白上货物的结合位点并定量测量它们的亲和力；2)确定接头与可溶性货物片段之间的复合体的晶体结构；3)利用突变分析将结构和功能联系起来。 适配器蛋白4(AP-4)是最近发现的、特征最差的异四聚体适配器蛋白(AP)复合体家族的成员，它介导后高尔基体隔室跨膜货物的分选。在上一财年，我们报道了阿尔茨海默病淀粉样前体蛋白(APP)胞浆尾部的YKFFE序列与AP-4的mu4亚单位的相互作用。生化和X射线结晶学分析表明，APP序列的性质和mu4上结合位点的位置与其他信号-适配器相互作用不同。APP-AP-4相互作用的中断减少了APP对内小体的定位，并增强了伽马分泌酶催化的APP对致病淀粉样β多肽的切割。这些发现表明，APP和AP-4参与了一种不同类型的信号-适配器相互作用，该相互作用介导了APP从跨高尔基网络(TGN)到内小体的运输，从而减少了蛋白质的淀粉样蛋白加工。在本财年，我们重点研究了AP复合体在极化细胞中的独特识别机制。

Molecular Recognition by Clathrin Adaptors The clathrin coat plays a ubiquitous and fundamental role in endocytosis and in endosomal sorting within the eukaryotic cell. Clathrin forms a cage that surrounds cargo-bearing vesicles, but clathrin itself does not directly bind to cargo. Cargo is sorted into clathrin-coated vesicles by adaptor proteins that physically bridge cargo and clathrin. The best-known general purpose adaptors are the heterotetrameric adaptor protein complexes (AP complexes) and the multimodular GGA adaptor proteins. The overall goals of this project are 1) to identify the binding sites for cargo on adaptor proteins and measure their affinities quantitatively; 2) to determine the crystal structures of complexes between adaptors and soluble cargo fragments; and 3) to relate structure and function using mutational analysis. Adaptor protein 4 (AP-4) is the most recently discovered and least well-characterized member of the family of heterotetrameric adaptor protein (AP) complexes that mediate sorting of transmembrane cargo in post-Golgi compartments. In the previous FY, we reported the interaction of an YKFFE sequence from the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) with the mu4 subunit of AP-4. Biochemical and X-ray crystallographic analyses reveal that the properties of the APP sequence and the location of the binding site on mu4 are distinct from those of other signal-adaptor interactions. Disruption of the APP-AP-4 interaction decreases localization of APP to endosomes and enhances gamma-secretase-catalyzed cleavage of APP to the pathogenic amyloid-beta peptide. These findings demonstrate that APP and AP-4 engage in a distinct type of signal-adaptor interaction that mediates transport of APP from the trans-Golgi network (TGN) to endosomes, thereby reducing amyloidogenic processing of the protein. In this FY we focused on unique recognition mechanisms of AP complexes in polarized cells.