ISOLATION AND CHARACTERIZATION OF FORMIN-ASSOCIATED PROTEIN COMPLEXES IN BUDDIN

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。形成蛋白是保守的肌动蛋白丝成核因子，在酵母中组装肌动蛋白线的极化阵列是特别需要的。目前还不清楚这些形成蛋白是如何在整个细胞中起作用的，或者它们是如何整合到酵母中调节细胞骨架组织的各种信号通路中的。许多formin相互作用蛋白已经被涉及，但许多这些结合伙伴是使用重组系统确定的，例如双杂交试验或细菌表达蛋白。为了确定内源性formin的结合伙伴，我们打算通过串联亲和纯化分离酵母formin，并对回收的样品进行质谱分析。为此，我们用cooh末端标记了编码形成蛋白的BNI1基因。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已经产生了富含Bni1p的样品。通过对这些的后续分析，可以通过纯化酵母中携带复杂成员突变的酵母，以及受不同条件干扰调节酵母细胞骨架极性的各种信号通路的酵母，来探测这些复合体的性质。

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.