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Autophagy pp 189-196 | Cite as

Studies of Receptor-Atg8 Interactions During Selective Autophagy

  • Christine Abert
  • Sascha MartensEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)

Abstract

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 words

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

Notes

Acknowledgments

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).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories (MFPL), Vienna Biocenter (VBC)University of ViennaViennaAustria

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