Advertisement

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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)

Abstract

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 

Notes

Acknowledgments

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.

References

  1. 1.
    Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741CrossRefGoogle Scholar
  2. 2.
    Johansen T, Lamark T (2011) Selective autophagy mediated by autophagic adapter proteins. Autophagy 7:279–296CrossRefGoogle Scholar
  3. 3.
    Khaminets A, Behl C, Dikic I (2016) Ubiquitin-dependent and independent signals in selective autophagy. Trends Cell Biol 26:6–16CrossRefGoogle Scholar
  4. 4.
    Rogov V, Dotsch V, Johansen T, Kirkin V (2014) Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy. Mol Cell 53:167–178CrossRefGoogle Scholar
  5. 5.
    Stolz A, Ernst A, Dikic I (2014) Cargo recognition and trafficking in selective autophagy. Nat Cell Biol 16:495–501CrossRefGoogle Scholar
  6. 6.
    Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, Overvatn A, Bjorkoy G, Johansen T (2007) p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282:24131–24145CrossRefGoogle Scholar
  7. 7.
    Kirkin V, Lamark T, Sou YS, Bjorkoy G, Nunn JL, Bruun JA, Shvets E, Mcewan DG, Clausen TH, Wild P, Bilusic I, Theurillat JP, Overvatn A, Ishii T, Elazar Z, Komatsu M, Dikic I, Johansen T (2009) A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. Mol Cell 33:505–516CrossRefGoogle Scholar
  8. 8.
    Wild P, Farhan H, Mcewan DG, Wagner S, Rogov VV, Brady NR, Richter B, Korac J, Waidmann O, Choudhary C, Dotsch V, Bumann D, Dikic I (2011) Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 333:228–233CrossRefGoogle Scholar
  9. 9.
    Thurston TL, Ryzhakov G, Bloor S, Von Muhlinen N, Randow F (2009) The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nat Immunol 10:1215–1221CrossRefGoogle Scholar
  10. 10.
    Newman AC, Scholefield CL, Kemp AJ, Newman M, Mciver EG, Kamal A, Wilkinson S (2012) TBK1 kinase addiction in lung cancer cells is mediated via autophagy of Tax1bp1/Ndp52 and non-canonical NF-kappaB signalling. PLoS One 7:e50672CrossRefGoogle Scholar
  11. 11.
    Lu K, Psakhye I, Jentsch S (2014) Autophagic clearance of polyQ proteins mediated by ubiquitin-Atg8 adaptors of the conserved CUET protein family. Cell 158:549–563CrossRefGoogle Scholar
  12. 12.
    Thurston TL, Wandel MP, Von Muhlinen N, Foeglein A, Randow F (2012) Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature 482:414–418CrossRefGoogle Scholar
  13. 13.
    Mandell MA, Jain A, Arko-Mensah J, Chauhan S, Kimura T, Dinkins C, Silvestri G, Munch J, Kirchhoff F, Simonsen A, Wei Y, Levine B, Johansen T, Deretic V (2014) TRIM proteins regulate autophagy and can target autophagic substrates by direct recognition. Dev Cell 30:394–409CrossRefGoogle Scholar
  14. 14.
    Lynch-Day MA, Klionsky DJ (2010) The Cvt pathway as a model for selective autophagy. FEBS Lett 584:1359–1366CrossRefGoogle Scholar
  15. 15.
    Suzuki K, Kondo C, Morimoto M, Ohsumi Y (2010) Selective transport of alpha-mannosidase by autophagic pathways: identification of a novel receptor, Atg34p. J Biol Chem 285:30019–30025CrossRefGoogle Scholar
  16. 16.
    Mancias JD, Pontano Vaites L, Nissim S, Biancur DE, Kim AJ, Wang X, Liu Y, Goessling W, Kimmelman AC, Harper JW (2015) Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis. Elife 4.  https://doi.org/10.7554/eLife.10308
  17. 17.
    Novak I, Kirkin V, Mcewan DG, Zhang J, Wild P, Rozenknop A, Rogov V, Lohr F, Popovic D, Occhipinti A, Reichert AS, Terzic J, Dotsch V, Ney PA, Dikic I (2010) Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep 11:45–51CrossRefGoogle Scholar
  18. 18.
    Liu L, Feng D, Chen G, Chen M, Zheng Q, Song P, Ma Q, Zhu C, Wang R, Qi W, Huang L, Xue P, Li B, Wang X, Jin H, Wang J, Yang F, Liu P, Zhu Y, Sui S, Chen Q (2012) Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nat Cell Biol 14:177–185CrossRefGoogle Scholar
  19. 19.
    Bhujabal Z, Birgisdottir AB, Sjottem E, Brenne HB, Overvatn A, Habisov S, Kirkin V, Lamark T, Johansen T (2017) FKBP8 recruits LC3A to mediate Parkin-independent mitophagy. EMBO Rep 18:947–961CrossRefGoogle Scholar
  20. 20.
    Khaminets A, Heinrich T, Mari M, Grumati P, Huebner AK, Akutsu M, Liebmann L, Stolz A, Nietzsche S, Koch N, Mauthe M, Katona I, Qualmann B, Weis J, Reggiori F, Kurth I, Hubner CA, Dikic I (2015) Regulation of endoplasmic reticulum turnover by selective autophagy. Nature 522:354–358CrossRefGoogle Scholar
  21. 21.
    Birgisdottir AB, Lamark T, Johansen T (2013) The LIR motif - crucial for selective autophagy. J Cell Sci 126:3237–3247PubMedGoogle Scholar
  22. 22.
    Noda NN, Ohsumi Y, Inagaki F (2010) Atg8-family interacting motif crucial for selective autophagy. FEBS Lett 584:1379–1385CrossRefGoogle Scholar
  23. 23.
    Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107–132CrossRefGoogle Scholar
  24. 24.
    Alemu EA, Lamark T, Torgersen KM, Birgisdottir AB, Larsen KB, Jain A, Olsvik H, Overvatn A, Kirkin V, Johansen T (2012) ATG8 family proteins act as scaffolds for assembly of the ULK complex: sequence requirements for LC3-interacting region (LIR) motifs. J Biol Chem 287:39275–39290CrossRefGoogle Scholar
  25. 25.
    Fu MM, Holzbaur EL (2014) Integrated regulation of motor-driven organelle transport by scaffolding proteins. Trends Cell Biol 24:564–574CrossRefGoogle Scholar
  26. 26.
    Olsvik HL, Lamark T, Takagi K, Larsen KB, Evjen G, Overvatn A, Mizushima T, Johansen T (2015) FYCO1 contains a C-terminally extended, LC3A/B-preferring LC3-interacting region (LIR) motif required for efficient maturation of autophagosomes during basal autophagy. J Biol Chem 290:29361–29374CrossRefGoogle Scholar
  27. 27.
    Pankiv S, Alemu EA, Brech A, Bruun JA, Lamark T, Overvatn A, Bjorkoy G, Johansen T (2010) FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. J Cell Biol 188:253–269CrossRefGoogle Scholar
  28. 28.
    Popovic D, Akutsu M, Novak I, Harper JW, Behrends C, Dikic I (2012) Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers. Mol Cell Biol 32:1733–1744CrossRefGoogle Scholar
  29. 29.
    Mcewan DG, Popovic D, Gubas A, Terawaki S, Suzuki H, Stadel D, Coxon FP, Miranda De Stegmann D, Bhogaraju S, Maddi K, Kirchof A, Gatti E, Helfrich MH, Wakatsuki S, Behrends C, Pierre P, Dikic I (2015) PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins. Mol Cell 57:39–54CrossRefGoogle Scholar
  30. 30.
    Genau HM, Huber J, Baschieri F, Akutsu M, Dotsch V, Farhan H, Rogov V, Behrends C (2015) CUL3-KBTBD6/KBTBD7 ubiquitin ligase cooperates with GABARAP proteins to spatially restrict TIAM1-RAC1 Signaling. Mol Cell 57:995–1010CrossRefGoogle Scholar
  31. 31.
    Kalvari I, Tsompanis S, Mulakkal NC, Osgood R, Johansen T, Nezis IP, Promponas VJ (2014) iLIR: a web resource for prediction of Atg8-family interacting proteins. Autophagy 10:913–925CrossRefGoogle Scholar
  32. 32.
    Von Muhlinen N, Akutsu M, Ravenhill BJ, Foeglein A, Bloor S, Rutherford TJ, Freund SM, Komander D, Randow F (2012) LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy. Mol Cell 48:329–342CrossRefGoogle Scholar
  33. 33.
    Ma P, Schwarten M, Schneider L, Boeske A, Henke N, Lisak D, Weber S, Mohrluder J, Stoldt M, Strodel B, Methner A, Hoffmann S, Weiergraber OH, Willbold D (2013) Interaction of Bcl-2 with the autophagy-related GABAA receptor-associated protein (GABARAP): biophysical characterization and functional implications. J Biol Chem 288:37204–37215CrossRefGoogle Scholar
  34. 34.
    Kaufmann A, Beier V, Franquelim HG, Wollert T (2014) Molecular mechanism of autophagic membrane-scaffold assembly and disassembly. Cell 156:469–481CrossRefGoogle Scholar
  35. 35.
    Habisov S, Huber J, Ichimura Y, Akutsu M, Rogova N, Loehr F, Mcewan DG, Johansen T, Dikic I, Doetsch V, Komatsu M, Rogov VV, Kirkin V (2016) Structural and functional analysis of a novel interaction motif within UFM1-activating enzyme 5 (UBA5) required for binding to ubiquitin-like proteins and ufmylation. J Biol Chem 291:9025–9041CrossRefGoogle Scholar
  36. 36.
    Rogov VV, Stolz A, Ravichandran AC, Rios-Szwed DO, Suzuki H, Kniss A, Lohr F, Wakatsuki S, Dotsch V, Dikic I, Dobson RC, Mcewan DG (2017) Structural and functional analysis of the GABARAP interaction motif (GIM). EMBO Rep 18:1382–1396CrossRefGoogle Scholar
  37. 37.
    Frank R, Overwin H (1996) SPOT synthesis. Epitope analysis with arrays of synthetic peptides prepared on cellulose membranes. Methods Mol Biol 66:149–169PubMedGoogle Scholar
  38. 38.
    Kramer A, Schneider-Mergener J (1998) Synthesis and screening of peptide libraries on continuous cellulose membrane supports. Methods Mol Biol 87:25–39PubMedGoogle Scholar
  39. 39.
    Lystad AH, Ichimura Y, Takagi K, Yang Y, Pankiv S, Kanegae Y, Kageyama S, Suzuki M, Saito I, Mizushima T, Komatsu M, Simonsen A (2014) Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures. EMBO Rep 15:557–565CrossRefGoogle Scholar
  40. 40.
    Skytte Rasmussen M, Mouilleron S, Kumar Shrestha B, Wirth M, Lee R, Bowitz Larsen K, Abudu Princely Y, O’reilly N, Sjottem E, Tooze SA, Lamark T, Johansen T (2017) ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8. Autophagy 13:834–853CrossRefGoogle Scholar
  41. 41.
    Johansen T, Birgisdottir AB, Huber J, Kniss A, Dotsch V, Kirkin V, Rogov VV (2017) Methods for studying interactions between Atg8/LC3/GABARAP and LIR-containing proteins. Methods Enzymol 587:143–169CrossRefGoogle Scholar

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

Personalised recommendations