Biophysical studies of LC3 family proteins

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.