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Tools for retargeting proteins within Aspergillus nidulans.

Subbulakshmi Suresh1,2, Leymaan Abdurehman1, Aysha H Osmani1

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Researchers adapted the GFP Binding Protein (GBP) system for Aspergillus nidulans, enabling precise relocalization of green fluorescent protein (GFP)-tagged proteins. New strains facilitate studying protein interactions and functions in fungi.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Mycology

Background:

  • Endogenous green fluorescent protein (GFP) tagging allows live cell microscopy of proteins in organisms like Aspergillus nidulans.
  • Existing GFP-tagging methods in A. nidulans are widely used but can be enhanced for more precise studies.
  • The GFP Binding Protein (GBP) system, a high-affinity llama antibody fragment against GFP, offers a novel tool for protein manipulation.

Purpose of the Study:

  • To adapt and implement the GBP system for targeted protein relocalization in Aspergillus nidulans.
  • To create new A. nidulans strains for studying protein interactions and cellular localization using GBP-mediated retargeting.
  • To validate the efficacy of GBP-tagging constructs for specific cellular compartments.

Main Methods:

  • Developed four base plasmid cassettes for generating gene replacement GBP-tagging constructs via fusion PCR.
  • Utilized fusion PCR and gene targeting to create A. nidulans strains with SPA10-GBP and Tom20-GBP gene replacements.
  • Tested the retargeting capabilities of SPA10-GBP to septa and Tom20-GBP to mitochondria.

Main Results:

  • Successfully generated SPA10-GBP and Tom20-GBP strains in A. nidulans.
  • SPA10-GBP effectively targeted GFP-tagged proteins to forming and mature septa.
  • Tom20-GBP demonstrated high retargeting capacity, relocating Nup49-GFP from nuclear pore complexes to the cytoplasm associated with mitochondria.
  • Relocation of Nup49-GFP resulted in cold sensitivity, similar to nup49 gene deletion.

Conclusions:

  • The developed cassette constructs and gene replacement strategies facilitate precise protein-protein linkages in fungi.
  • The new SPA10-GBP and Tom20-GBP A. nidulans strains provide powerful tools for modulating and studying other GFP-tagged proteins.
  • This system enhances experimental approaches for investigating protein function and localization in filamentous fungi.