Learn more about GABARAP
T. Johansen, ... V.V. Rogov, in Methods in Enzymology, 2017
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.
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