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.
Keywords: Atg19; Atg8; Autophagy; Cargo receptor; Cytoplasm-to-vacuole targeting pathway; Fluorescence microscopy; In vitro reconstitution; Protein-protein interaction; Quantification; Selective autophagy.