Abstract
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.
Highlights
Having the ability to experimentally link proteins to each other within cells provides several experimental avenues to interrogate protein function
By providing an artificial chromatin-nuclear pore complexes (NPCs) bridge we were able to show that NPCs were segregated to daughter nuclei in the absence of Nup2, without this link NPCs are mis-segregated into the cytoplasm after mitosis [1]
We report the generation of strains (Table 1, Strains used in this study) with GFP Binding Protein (GBP) protein fusions to enable retargeting of green fluorescent protein (GFP)-tagged proteins to either septa or mitochondria
Summary
Having the ability to experimentally link proteins to each other within cells provides several experimental avenues to interrogate protein function. If the linked target proteins locate stably to specific structures it is possible to experimentally bridge the structures to which the two proteins belong Using this approach we recently provided linkage between nuclear pore complexes (NPCs) and nuclear chromatin in the model fungus Aspergillus nidulans [1]. The GFP-GBP system has been developed for use in human cells and has been successfully employed in plants, fission yeast and prokaryotes but has had limited application in filamentous fungi [3,4,5,6] This system is based on a genetically encoded Llama single chain antibody against GFP [2, 3]. GBP recognizes GFP and YFP but not CFP or DsRed derivatives, such as mCherry or mRFP [2]
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