ISOLATION AND CHARACTERIZATION OF FORMIN-ASSOCIATED PROTEIN COMPLEXES IN BUDDING

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 formins是保守的肌动蛋白亲核因子，并且在酵母中特别需要组装肌动蛋白电缆的极化阵列。 目前还不清楚这些formins如何在整个细胞中发挥作用，或者它们如何整合到调节酵母细胞骨架组织的各种信号通路中。 许多与福尔马林相互作用的蛋白质都受到影响，但其中许多结合伴侣是使用重组系统（例如双杂交测定或细菌表达的蛋白质）鉴定的。 为了鉴定内源性formin的结合伴侣，我们打算通过串联亲和纯化分离酵母菌，并将回收的样品进行质谱分析。 为此，我们对编码formin的BNI 1基因进行了COOH末端标记。基于遗传相互作用和细胞骨架和细胞形态，蛋白质是功能性的。 正在进行的初步纯化产生了高度富集Bni 1 p的样品。 通过对这些的后续分析，可以通过从携带复合物成员突变的酵母以及经受干扰调节酵母中细胞骨架极性的各种信号传导途径的不同条件的酵母中纯化来探测回收的复合物的性质。

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.