title: Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins.
abstract: LC3/GABARAP proteins (LC3/GABARAPs) are mammalian orthologues of yeast Atg8, small ubiquitin (Ub)-like proteins (UBLs) whose covalent attachment to lipid membranes is crucial for the growth and closure of the double membrane vesicle called the autophagosome. In the past decade, it was demonstrated that Atg8/LC3/GABARAPs are also required for autophagic degradation of cargos in a selective fashion. Cargo selectivity is ensured by receptor proteins, such as p62/SQSTM1, NBR1, Cue5, Atg19, NIX, Atg32, NCOA4, and FAM134B, which simultaneously bind Atg8/LC3/GABARAPs and the cargo together, thereby linking the core autophagic machinery to the target structure: a protein, an organelle, or a pathogen. LC3-interacting regions (LIRs) are short linear motifs within selective autophagy receptors and some other structural and signaling proteins (e.g., ULK1, ATG13, FIP200, and Dvl2), which mediate binding to Atg8/LC3/GABARAPs. Identification and characterization of LIR-containing proteins have provided important insights into the biology of the autophagy pathway, and studying their interactions with the core autophagy machinery represents a growing area of autophagy research. Here, we present protocols for the identification of LIR-containing proteins, i.e., by yeast-two-hybrid screening, glutathione S-transferase (GST) pulldown experiments, and peptide arrays. The use of two-dimensional peptide arrays also represents a powerful method to identify the residues of the LIR motif that are critical for binding. We also describe a biophysical method for studying interactions between Atg8/LC3/GABARAP and LIR-containing proteins and a protocol for preparation and purification of LIR peptides.
authors.affiliation: Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø-The Arctic University of Norway, Tromsø, Norway. Electronic address: terje.johansen@uit.no.
authors.forename: T
authors.initials: T
authors.lastname: Johansen
authors.affiliation: Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø-The Arctic University of Norway, Tromsø, Norway.
authors.forename: Å B
authors.initials: ÅB
authors.lastname: Birgisdottir
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt am Main, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Huber
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt am Main, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Kniss
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt am Main, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Dötsch
authors.affiliation: Merck KGaA, Darmstadt, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Kirkin
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt am Main, Germany. Electronic address: rogov@bpc.uni-frankfurt.de.
authors.forename: V V
authors.initials: VV
authors.lastname: Rogov
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D051017:Apoptosis Regulatory Proteins; D000071190:Autophagy-Related Protein 8 Family / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D020798:Two-Hybrid System Techniques
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D000071190:Autophagy-Related Protein 8 Family; C116741:GABARAP protein, human; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins
keywords: Atg8; GABARAP; GST pulldown; ITC; LC3; LIR; Peptide array; UBL; Yeast-two-hybrid