ISOLATION AND CHARACTERIZATION OF FORMIN-ASSOCIATED PROTEIN COMPLEXES IN BUDDING

出芽过程中福尔马林相关蛋白复合物的分离和表征

• 批准号: 8171376
• 负责人: Anthony P. Bretscher
• 金额: $ 0.24万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171376
• 关键词: Actins; Affinity Chromatography; Binding; Cells; Cellular Morphology; Complex; Computer Retrieval of Information on Scientific Projects Database; Cytoskeletal Modeling; Funding; Genes; Genetic; Grant; Institution; Mass Spectrum Analysis; Microfilaments; Mutation; Nature; Proteins; Recombinants; Research; Research Personnel; Resources; Sampling; Signal Pathway; Source; System; Two-Hybrid System Techniques; United States National Institutes of Health; Yeasts; base; member; protein complex

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The formins are conserved actin filament-nucleating factors, and are required specifically in yeast to assemble polarized arrays of actin cables. It is not yet understood how these formins function within the context of the whole cell, or how they are integrated into the various signaling pathways that regulate cytoskeletal organization in yeast. A number of formin-interacting proteins have been implicated, but many of these binding partners were identified using recombinant systems, such as two-hybrid assays or bacterially-expressed proteins. To identify binding partners for the endogenous formins, we intend to isolate a yeast formin through tandem-affinity purification, and subject the recovered samples to mass spectroscopy. To that end, we have COOH-terminally tagged the formin-encoding BNI1 gene. Based on genetic interactions and cytoskeletal and cell morphology, the protein is functional. Preliminary purifications underway have yielded samples highly enriched for Bni1p. With subsequent analysis of these, the nature of the complexes recovered can be probed through purifications from yeast bearing mutations of complex members, as well as yeast subjected to varying conditions that perturb the various signalling pathways that modulate cytoskeletal polarity in yeast.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 造型是保守的肌动蛋白丝 - 核因子，特别是在酵母中需要组装肌动蛋白电缆的极化阵列。 尚不理解这些formins在整个细胞的背景下如何发挥作用，或者如何将它们整合到调节酵母中细胞骨架组织的各种信号通路中。 已经涉及许多形式的相互作用蛋白，但是使用重组系统（例如两种杂交测定法或细菌表达的蛋白质）鉴定了许多这些结合伴侣。 为了鉴定内源性造型的结合伴侣，我们打算通过串联纯度纯化分离酵母菌，并将回收的样品对质量光谱法进行。 为此，我们用COOH末端标记了编码的BNI1基因。基于遗传相互作用以及细胞骨架和细胞形态，该蛋白质功能性。 正在进行的初步净化已产生了高度富含BNI1P的样品。 通过对这些分析的后续分析，可以通过从复杂成员的酵母轴承突变以及经历不同条件的酵母中进行纯化来探测所回收的络合物的性质，这些酵母菌会扰动各种信号传导途径调节酵母中细胞骨骼极性的各种信号传导途径。