PT  - JOURNAL ARTICLE
AU  - Zhong-Qiu Yu
AU  - Xiao-Man Liu
AU  - Dan Zhao
AU  - Dan-Dan Xu
AU  - Li-Lin Du
TI  - Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay
AID  - 10.1101/2021.04.13.439745
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.04.13.439745
4099  - http://biorxiv.org/content/early/2021/04/14/2021.04.13.439745.short
4100  - http://biorxiv.org/content/early/2021/04/14/2021.04.13.439745.full
AB  - 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 StatementThe authors have declared no competing interest.Y2Hyeast two-hybridFRETfluorescence resonance energy transferBiFCbimolecular fluorescence complementationAIMAtg8-family-interacting motifNLSnuclear localization signal