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，并将其应用于研究靶向膜交通囊泡载体的多亚基拴系复合物（MTCs）。 (1)我们最近开发了“单层纯化”和“亲和网格”技术，其使用Ni-NTA脂质在脂质单层中直接从细胞提取物中招募His标记的靶蛋白。我们建议扩展亲和网格库，以包括捕获组氨酸以外的标签的蛋白质。特别是，我们将测试生物素化脂质使用抗生物素蛋白衔接子募集蛋白质，合成具有谷胱甘肽（D，l-谷氨酰基-l-半胱氨酰甘氨酸，GSH）基团的脂质以募集具有谷胱甘肽-S-转移酶（GST）标签的蛋白质，以及亲和标记的Fc片段以募集具有串联亲和纯化（TAP）标签的蛋白质。由于TAP标记的和GST融合构建体的酵母文库的商业可用性，标记的Fc片段和GSH功能化的脂质可以允许亲和网格的高通量应用。 (2)我们将继续我们的多亚基拴系复合物（MTCs）的结构研究，利用亲和网格的方法。MTC介导转运囊泡与其靶膜之间的第一次接触，并且被认为通过与Rab GTP酶、外壳蛋白和SNARE的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的结构和DsH复合物和COG的Cog 1 -4子复合物。通过确定其他MTC的结构- TRAPPIII，HOPS，GARP，完整的COG和外囊-并分析它们与Rab GTP酶和SNARE蛋白的相互作用，我们的目标是了解这些MTC是如何组织的，它们的不同组织如何介导囊泡束缚，什么样的构象变化是MTC辅助SNARE复合物组装的基础，以及亚基突变如何干扰MTC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.