Project 2: Walz

说明)： 该计划项目的这一组成部分将增强我们为单粒子电子显微镜(EM)开发的一种新的样品制备方法，并将其应用于研究以膜交通的囊泡载体为目标的多亚单位系留复合体(MTCs)。 (1)我们最近发展了“单层纯化”和“亲和网格”技术，利用脂质单层中的Ni-NTA脂类直接从细胞提取液中招募His标记的靶蛋白。我们建议扩展亲和网格曲目，以包括捕获带有组氨酸以外标签的蛋白质。特别是，我们将测试生物素化的脂类以招募使用亲和素接头的蛋白质，合成含有谷胱甘肽(D，L-谷氨酰-L-半胱氨酸甘氨酸)基团的脂质以招募带有谷胱甘肽-S转移酶标签的蛋白质，以及使用亲和标签的Fc片段招募带有串联亲和纯化标签的蛋白质。由于TAP标记的酵母文库和GST融合构建的酵母文库的商业可用性，标记的Fc片段和GSH功能化的脂质可能允许亲和网格的高通量应用。 (2)我们将继续利用亲和网格方法对多亚单位系留复合体(MTCs)的结构进行研究。MTCs介导运输囊泡与其靶膜之间的第一次接触，并被认为通过与Rab GTP酶、外壳蛋白和SNARs相互作用来协调囊泡捕获、对接和融合。我们已经获得了TRAPPI和II的结构以及COG的DSH络合物和Cog1-4亚络合物的结构。通过确定额外的MTC的结构- TRAPPIII、HOPS、GARP、完整COG和胞外--并分析它们与Rab GTP酶和SNARE蛋白的相互作用，我们的目的是了解这些MTCs是如何组织的，它们的不同组织如何介导囊泡拴系，MTC辅助的SNARE复合体组装背后的构象变化，以及亚单位的突变如何干扰MTCs的功能。

instructions): This component of the Program Project will enhance a new sample preparation method we have developed for single-particle electron microscopy (EM) and apply it to studying the multisubunit tethering complexes (MTCs) that target vesicular carriers of membrane traffic. (1) We have recently developed the "monolayer purification" and "Affinity Grid" techniques, which use Ni-NTA lipids in a lipid monolayer to recruit His-tagged target proteins directly from cell extracts. We propose to extend the Affinity Grid repertoire to include capture of proteins with tags other than histidine. In particular, we will test biotinylated lipids to recruit proteins using an avidin adaptor, synthesis of a lipid with a glutathione (D,l-glutamyl-l-cysteinylglycine, GSH) group to recruit proteins with a glutathione-S-transferase (GST) tag, and affinity-tagged Fc fragments to recruit proteins with a tandem affinity purification (TAP) tag. Because of the commercial availability of yeast libraries of TAP-tagged and GST-fusion constructs, the tagged Fc fragments and the GSH-functionalized lipid may allow high-throughput applications of the Affinity Grid. (2) We will continue our structural studies of multisubunit tethering complexes (MTCs), taking advantage of the Affinity Grid approach. MTCs mediate the first contact between a transport vesicle and its target membrane and are thought to orchestrate vesicle capture, docking, and fusion through interactions with Rab GTPases, coat proteins and SNAREs. We have already obtained structures of TRAPPI and II and of the DsH complex and the Cog1-4 subcomplex of COG. By determining the structures of additional MTCs - TRAPPIII, HOPS, GARP, intact COG and exocyst - and analyzing their interactions with Rab GTPases and SNARE proteins, we aim to understand how these MTCs are organized, how their different organizations mediate vesicle tethering, what conformational changes underlie MTC-assisted SNARE complex assembly, and how mutations in subunits interfere with function of MTCs.