Studies of Receptor-Atg8 Interactions During Selective Autophagy
Autophagy research frequently requires the determination of protein-protein interactions. The experimental system described in this chapter allows a simple, versatile, and quantitative in vitro analysis of interactions between recombinant cargo receptor and Atg8 proteins by fluorescence microscopy. The assay can be easily modified to study other protein-protein interactions. The purified autophagy receptor is recruited to affinity resins via a suitable tag and then added to fluorescently labeled ATG8 in solution. The relative strength of the interaction can be assessed by determination of the fluorescence intensity on the surface of the bead at an equilibrium binding state. Thereby different interaction partners can be quantitatively compared, and weak or interactions with high off rates can be detected and quantified.
Key wordsAutophagy Selective autophagy Cytoplasm-to-vacuole targeting pathway Cargo receptor Atg8 Atg19 Protein-protein interaction In vitro reconstitution Fluorescence microscopy Quantification
We thank Justyna Sawa-Makarska and Verena Baumann for comments on the manuscript. Christine Abert is supported by a Doc fellowship of the Austrian Academy of Sciences (ÖAW).
- 4.Sawa-Makarska J, Abert C, Romanov J, Zens B, Ibiricu I, Martens S (2014) Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane-cargo apposition during selective autophagy. Nat Cell Biol 16(5):425–433. https://doi.org/10.1038/ncb2935CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Fracchiolla D, Sawa-Makarska J, Zens B, Ruiter A, Zaffagnini G, Brezovich A, Romanov J, Runggatscher K, Kraft C, Zagrovic B, Martens S (2016) Mechanism of cargo-directed Atg8 conjugation during selective autophagy. Elife 5:e18544. https://doi.org/10.7554/eLife.18544CrossRefPubMedPubMedCentralGoogle Scholar
- 10.von Stetten D, Noirclerc-Savoye M, Goedhart J, Gadella TW Jr, Royant A (2012) Structure of a fluorescent protein from Aequorea victoria bearing the obligate-monomer mutation A206K. Acta Crystallogr Sect F Struct Biol Cryst Commun 68(Pt 8):878–882. https://doi.org/10.1107/S1744309112028667CrossRefGoogle Scholar