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Autophagy pp 91-117 | Cite as

Biophysical Studies of LC3 Family Proteins

  • Javier H. Hervás
  • Zuriñe Antón
  • Alicia AlonsoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)

Abstract

Autophagy is an important cellular process in which cell components are degraded in a controlled way and their building blocks are recycled into new macromolecules. Autophagy starts within a double-membrane container, the autophagosome, itself the result of a number of interconversions of cell membranous elements. In our recent work, we have described reconstituted model systems for the interactions of autophagy proteins with membrane lipid bilayers and for the autophagy protein-mediated vesicle tethering and fusion, with the aim of ultimately reconstituting the autophagosome formation. The present chapter describes in detail (a) the steps required for the preparation of semisynthetic lipid vesicles (liposomes), including giant unilamellar vesicles, (b) ultracentrifugation and fluorescence methods for assaying protein binding to membranes, and (c) procedures for assessing vesicle–vesicle aggregation and fusion. The latter include methods for intervesicular total lipid mixing, mixing of lipids in the vesicle inner monolayers, and aqueous contents mixing.

Key words

LC3/GABARAP proteins Autophagy proteins Membrane fusion Liposomes Fusion assays Membrane biophysics Protein–lipid interactions 

Notes

Acknowledgments

The authors thank Ms. Araceli Marcos for her technical support. This work was supported in part by grants from FEDER/Spanish Ministry of Economy (BFU 2011-28566, BFU 2015-66306-P) and the Basque Government (IT838-13). J.H.H. and Z.A. were predoctoral fellows supported by the University of the Basque Country; J.H.H. is a postdoctoral fellow supported by the University of the Basque Country, and Z.A. is a postdoctoral research associate supported by BrisSynBio at the University of Bristol.

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Copyright information

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

Authors and Affiliations

  • Javier H. Hervás
    • 1
    • 2
  • Zuriñe Antón
    • 1
    • 2
  • Alicia Alonso
    • 1
    • 2
    Email author
  1. 1.Instituto Biofisika (CSIC, UPV/EHU)BilbaoSpain
  2. 2.Departamento de Bioquímica y Biología MolecularUniversidad del País VascoBilbaoSpain

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