Abstract：
Targeting a protein of interest to a subcellular location by linking it to another protein is a commonly used approach to help determine function in many model systems. Such targeting strategies rely on the creation of functional protein-protein fusions followed by microscopic examination if one or both proteins have fluorescent tags. In this paper, using the model filamentous fungus Aspergillus nidulans, we describe methods to link GFP-tagged proteins to other proteins in the cell by fusing the latter with a GFP-Binding Protein (GBP) that has a high affinity for GFP. This method enables rapid generation of strains with linked proteins in filamentous fungi by sexual crossing or transformations. Additionally, if these two linked proteins stably associate with subcellular structures, it is possible to link the structures using this approach. For example, we used this method to link Nuclear Pore Complexes (NPCs) with mitotic chromatin in A. nidulans. This was done to show that the NPC protein Nup2, that uniquely transitions from NPC onto mitotic chromatin, couples NPC segregation with chromatin segregation by bridging these two structures. In the absence of Nup2, we used the described approach to show that an artificial NPC-chromatin bridge was sufficient for faithful NPC segregation.
摘要：
通过将感兴趣的蛋白质与另一种蛋白质连接而将其靶向亚细胞位置是帮助确定许多模型系统中的功能的常用方法。这种靶向策略依赖于功能性蛋白质-蛋白质融合体的产生,随后如果一个或两个蛋白质具有荧光标签,则进行显微镜检查。在本文中,使用丝状真菌构巢曲霉模型，我们描述了通过将GFP-标记的蛋白与对GFP具有高亲和力的GFP-结合蛋白(GBP)融合而将后者连接到细胞中的其他蛋白的方法。该方法能够通过有性杂交或转化在丝状真菌中快速产生具有连接蛋白的菌株。此外,如果这两种连接的蛋白质与亚细胞结构稳定结合，使用这种方法可以连接结构。例如,我们使用这种方法将核孔隙复合物（NPCs）与有丝分裂染色质联系起来。这样做是为了表明NPC蛋白Nup2从NPC独特地过渡到有丝分裂染色质，通过桥接这两个结构将NPC分离与染色质分离结合起来。在没有Nup2的情况下，我们使用所描述的方法来表明人工NPC-染色质桥足以进行忠实的NPC分离。
