Tools for retargeting proteins within Aspergillus nidulans

Endogenously tagging proteins with green fluorescent protein (GFP) enables the visualization of the tagged protein using live cell microscopy. GFP-tagging is widely utilized to study biological processes in model experimental organisms including filamentous fungi such as Aspergillus nidulans. Many strains of A. nidulans have therefore been generated with different proteins endogenously tagged with GFP. To further enhance experimental approaches based upon GFP-tagging, we have adapted the GFP Binding Protein (GBP) system for A. nidulans. GBP is a genetically encoded Llama single chain antibody against GFP which binds GFP with high affinity. Using gene replacement approaches, it is therefore possible to link GBP to anchor proteins, which will then retarget GFP-tagged proteins away from their normal location to the location of the anchor-GBP protein. To facilitate this approach in A. nidulans, we made four base plasmid cassettes that can be used to generate gene replacement GBP-tagging constructs by utilizing fusion PCR. Using these base cassettes, fusion PCR, and gene targeting approaches, we generated strains with SPA10-GBP and Tom20-GBP gene replacements. These strains enabled test targeting of GFP-tagged proteins to septa or to the surface of mitochondria respectively. SPA10-GBP is shown to effectively target GFP-tagged proteins to both forming and mature septa. Tom20-GBP has a higher capacity to retarget GFP-tagged proteins being able to relocate all Nup49-GFP from its location within nuclear pore complexes (NPCs) to the cytoplasm in association with mitochondria. Notably, removal of Nup49-GFP from NPCs causes cold sensitivity as does deletion of the nup49 gene. The cassette constructs described facilitate experimental approaches to generate precise protein-protein linkages in fungi. The A. nidulans SPA10-GBP and Tom20-GBP strains can be utilized to modulate other GFP-tagged proteins of interest.

利用绿色荧光蛋白（GFP）对蛋白质进行内源标记，可通过活细胞显微成像技术直观观测被标记的蛋白质。GFP标记技术被广泛应用于模式实验生物的生物学过程研究，涵盖丝状真菌等模式生物，其中包括构巢曲霉（Aspergillus nidulans）。因此，科研人员已构建诸多携带不同内源GFP标记蛋白质的构巢曲霉菌株。为进一步优化基于GFP标记的实验策略，我们针对构巢曲霉改造了GFP结合蛋白（GBP）系统。GFP结合蛋白是一种经遗传编码的羊驼单链抗体，可与GFP发生高亲和力结合。借助基因替换技术，可将GBP与锚定蛋白相连，随后该锚定蛋白-GBP融合蛋白即可将GFP标记的蛋白质从其天然定位处，重新靶向至锚定蛋白的所在位置。为在构巢曲霉中推广该实验方法，我们构建了4种基础质粒盒，可通过融合PCR技术制备用于基因替换的GBP标记构建体。利用上述基础质粒盒、融合PCR及基因靶向技术，我们成功构建了携带SPA10-GBP与Tom20-GBP基因替换的构巢曲霉菌株。这两款菌株可分别实现将GFP标记蛋白靶向至真菌隔膜或线粒体表面。实验结果表明，SPA10-GBP可高效将GFP标记蛋白靶向至新生及成熟隔膜。Tom20-GBP具备更强的GFP标记蛋白重靶向能力，可将所有定位于核孔复合体（NPCs）内的Nup49-GFP，转移至与线粒体相关的细胞质区域。值得注意的是，将Nup49-GFP从核孔复合体中移除会导致菌株出现冷敏感表型，该表型与nup49基因敲除所引发的表型一致。本文所述的质粒盒载体可助力真菌中精准蛋白质间相互连接的实验研究。构巢曲霉SPA10-GBP与Tom20-GBP菌株可用于调控其他目标GFP标记蛋白。