Molecular Recognition by Clathrin Adaptors

网格蛋白适配器的分子识别

• 批准号: 8148745
• 负责人: James Hurley
• 金额: $ 27.25万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148745
• 关键词: Clathrin Adaptors; molecular recognition

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. An YKFFE sequence from the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) interacts with the μ4 subunit of AP-4. X-ray crystallographic analyses revealed that the properties of the APP sequence and the location of the binding site on μ4 are distinct from those of other signal-adaptor interactions. The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs.

网格蛋白衔接子的分子识别 网格蛋白外壳在真核细胞内的内吞作用和内体分选中起着普遍存在的和基本的作用。网格蛋白形成一个笼，围绕货物轴承囊泡，但网格蛋白本身不直接结合货物。货物通过衔接蛋白被分选到网格蛋白包被的囊泡中，衔接蛋白物理桥接货物和网格蛋白。最著名的通用衔接子是异源四聚体衔接子蛋白复合物（AP复合物）和多模块GGA衔接子蛋白。该项目的总体目标是：1）鉴定衔接蛋白上货物的结合位点并定量测量其亲和力; 2）确定衔接蛋白和可溶性货物片段之间复合物的晶体结构; 3）使用突变分析将结构和功能联系起来。 衔接蛋白4（AP-4）是最近发现的并且最不充分表征的异源四聚体衔接蛋白（AP）复合物家族的成员，其介导高尔基体后区室中跨膜货物的分选。来自阿尔茨海默病淀粉样前体蛋白（APP）的胞质尾部的YKFFE序列与AP-4的4亚基相互作用。X射线晶体学分析表明，APP序列的性质和4上的结合位点的位置与其他信号-适配器相互作用的性质不同。 Hermansky-Pudlak综合征（HPS）是一种遗传性色素减退和出血性疾病，由溶酶体相关细胞器（LRO）如黑素体和血小板致密体的生物合成缺陷引起。HPS由人类8个基因和小鼠16个基因中的任何一个突变引起。其中两个基因，HPS 1和HPS 4，编码溶酶体相关细胞器复合物-3（BLOC-3）的生物发生组分。在此，我们表明，在昆虫细胞中产生的重组HPS 1-HPS 4可以有效地分离为1：1异二聚体。分析超离心表明，该复合物的分子量为146 kDa，相当于天然复合物的分子量和HPS 1和HPS 4的预测分子量之和。这表明HPS 1和HPS 4在没有任何其他蛋白作为BLOC-3的一部分的情况下直接相互作用。有限的蛋白水解和缺失分析表明，这两个亚基相互作用，在整个大部分的长度与唯一的例外的长，非结构化的循环在中央部分的HPS 4。相互作用筛选揭示了BLOC-3与GTP结合形式的内体GTP酶Rab 9的特异性和强相互作用。这种相互作用由HPS 4和Rab 9的开关I和II区域介导。这些特征表明BLOC-3可能在LRO的生物发生中起Rab 9效应子的作用。