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Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay

View ORCID ProfileZhong-Qiu Yu, View ORCID ProfileXiao-Man Liu, View ORCID ProfileDan Zhao, Dan-Dan Xu, View ORCID ProfileLi-Lin Du
doi: https://doi.org/10.1101/2021.04.13.439745
Zhong-Qiu Yu
1National Institute of Biological Sciences, 102206 Beijing, China
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Xiao-Man Liu
1National Institute of Biological Sciences, 102206 Beijing, China
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Dan Zhao
1National Institute of Biological Sciences, 102206 Beijing, China
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Dan-Dan Xu
1National Institute of Biological Sciences, 102206 Beijing, China
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Li-Lin Du
1National Institute of Biological Sciences, 102206 Beijing, China
2Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, 102206 Beijing, China
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  • For correspondence: dulilin@nibs.ac.cn
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Abstract

Protein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeast Schizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins, transmembrane proteins, and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.

Competing Interest Statement

The authors have declared no competing interest.

  • Abbreviations

    Y2H
    yeast two-hybrid
    FRET
    fluorescence resonance energy transfer
    BiFC
    bimolecular fluorescence complementation
    AIM
    Atg8-family-interacting motif
    NLS
    nuclear localization signal
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    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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    Posted April 14, 2021.
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    Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay
    Zhong-Qiu Yu, Xiao-Man Liu, Dan Zhao, Dan-Dan Xu, Li-Lin Du
    bioRxiv 2021.04.13.439745; doi: https://doi.org/10.1101/2021.04.13.439745
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    Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay
    Zhong-Qiu Yu, Xiao-Man Liu, Dan Zhao, Dan-Dan Xu, Li-Lin Du
    bioRxiv 2021.04.13.439745; doi: https://doi.org/10.1101/2021.04.13.439745

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