ISOLATION AND CHARACTERIZATION OF FORMIN-ASSOCIATED PROTEIN COMPLEXES IN BUDDING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 福尔马林是保守的肌动蛋白细丝成核因子，在酵母中是组装极化的肌动蛋白电缆阵列所必需的。目前还不清楚这些福尔马林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。一些与福尔明相互作用的蛋白质已经被牵连，但这些结合伙伴中的许多是通过重组系统鉴定的，例如双杂交试验或细菌表达的蛋白质。为了确定内源福尔敏的结合伙伴，我们打算通过串联亲和纯化分离酵母福尔明，并对回收的样品进行质谱分析。为此，我们对编码Forin的BNI1基因进行了COOH末端标记。根据遗传相互作用以及细胞骨架和细胞形态，该蛋白质具有功能。正在进行的初步净化已经产生了高度浓缩的Bni1p样本。通过随后对这些进行分析，可以通过从携带复杂成员突变的酵母以及在不同条件下扰乱酵母中调节细胞骨架极性的各种信号通路的酵母中提纯，来探索回收的复合体的性质。

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.