Protein Retargeting in Aspergillus nidulans to Study the Function of Nuclear Pore Complex Proteins.

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.