Molecular Recognition by Clathrin Adaptors

网格蛋白适配器的分子识别

• 批准号: 7967359
• 负责人: James Hurley
• 金额: $ 28.82万
• 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/7967359
• 关键词: Adaptor Signaling Protein; Affinity; Binding; Binding Sites; Biological Assay; C-terminal; Cells; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Complex; Endocytosis; Eukaryotic Cell; Family; Goals; Human; In Vitro; Integral Membrane Protein; Laboratories; Link; Measures; Membrane; N-terminal; Names; Pathway interactions; Play; Proteins; Role; Shapes; Signal Transduction; Sorting - Cell Movement; Structure; Vesicle; Yeasts; golgi associated, gamma adaptin homologous, ADP-ribosylation factor interacting protein; in vivo; member; molecular recognition; protein complex; receptor; scaffold

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. The subunits of the AP complexes and the GGAs define conserved structural domains that are also found in other endocytic proteins. In the past year, we collaborated with Beverly Wendland (JHU) to define the structures of the domains of the endocytic protein Syp1. Syp1 is proposed to be a founding member of the newly-named muniscin family of endocytic adaptors that is conserved from yeast to humans. Solving the structures of yeast muniscin domains confirmed the unique combination of an N-terminal domain homologous to the crescent-shaped membrane tubulating EFC/F-BAR domains and a C-terminal domain homologous to cargo-binding mu homology domains (muHD). In vitro and in vivo assays in the Wendland laboratory confirmed membrane tubulation activity for muniscin EFC/F-BAR domains. The mHD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. A transmembrane protein previously implicated in polarized Rho1 signaling was identified as a cargo of the yeast adaptor protein. We propose that muniscins directly link cargo selection with vesicle formation, thereby regulating receptors that stimulate cell polarization pathways.

网格蛋白适配器的分子识别 网格蛋白涂层在内吞作用和真核生物细胞内的内体分选中起普遍存在的基本作用。网状蛋白形成围绕载有货物的囊泡的笼子，但网状蛋白本身并未直接与货物结合。货物通过物理桥接货物和网格蛋白的衔接蛋白将货物分类。最著名的通用适配器是异晶衔接蛋白复合物（AP复合物）和多模块化GGA适配器蛋白。该项目的总体目标是1）确定适配器蛋白上货物的结合位点并定量测量其亲和力； 2）确定适配器和可溶性货物碎片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。 AP复合物和GGA的亚基定义了在其他内吞蛋白中也发现的保守结构结构域。在过去的一年中，我们与Beverly Wendland（JHU）合作定义了内吞蛋白SYP1领域的结构。有人认为SYP1是新命名的Muniscin家族的内吞转移家族的创始成员，从酵母到人类。解决酵母Muniscin结构域的结构证实了与新月形膜管EFC/F-BAR结构域同源的N末端结构域的独特组合，以及与货物结合的MU同源性领域（MUHD）同源的C端结构域。温德兰实验室中的体外和体内测定证实了Muniscin EFC/F-BAR结构域的膜管活性。 MHD结构域与内吞适配器/支架EDE1/EPS15保持了保守的相互作用，这会影响Muniscin定位。先前与极化Rho1信号传导的跨膜蛋白被确定为酵母衔接蛋白的货物。我们建议Muniscins将货物选择与囊泡形成联系起来，从而调节刺激细胞极化途径的受体。