Autophagy pp 149-161 | Cite as

Use of Peptide Arrays for Identification and Characterization of LIR Motifs

  • Mads Skytte Rasmussen
  • Åsa Birna Birgisdottir
  • Terje JohansenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)


The mammalian ATG8 proteins (LC3A-C/GABARAP, GABARAPL1, and GABARAPL2) are small ubiquitin-like proteins critically involved in macroautophagy. Their processed C-termini are posttranslationally conjugated to a phosphatidylethanolamine moiety, enabling their insertion into the lipid bilayers of both the inner and outer membranes of the forming autophagosomes. The ATG8s bind a diverse selection of proteins including cargo receptors for selective autophagy, members of the core autophagy machinery, and other proteins involved in formation, transport, and maturation (fusion to lysosomes) of autophagosomes. Protein binding to the ATG8s is in most cases mediated by short, conserved sequence motifs known as LC3-interacting regions (LIRs). Here, we present a protocol for identifying putative LIR motifs in a whole protein sequence using peptide arrays generated by SPOT synthesis on nitrocellulose membranes. The use of two-dimensional peptide arrays allows for further identification of specific residues critical for LIR binding.

Key words

Autophagy Atg8 GABARAP LC3 LIR Peptide array 



We are extremely grateful to Ola Rumohr Blingsmo at the Centre for Molecular Medicine Norway, NCMM-Administration and Core Facilities (NCMM ADMIN), Faculty of Medicine, University of Oslo, for advice and synthesizing the peptide arrays. The technical assistance of Gry Evjen is greatly appreciated. This work was funded by grants from the FRIBIO and FRIBIOMED programs of the Norwegian Research Council (grant numbers 196898 and 214448) and the Norwegian Cancer Society (grant number 71043-PR-2006-0320) to T.J.


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Copyright information

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

Authors and Affiliations

  • Mads Skytte Rasmussen
    • 1
  • Åsa Birna Birgisdottir
    • 1
  • Terje Johansen
    • 1
    Email author
  1. 1.Molecular Cancer Research Group, Department of Medical BiologyUniversity of Tromsø – The Arctic University of NorwayTromsøNorway

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