In Vitro Reconstitution of Atg8 Conjugation and Deconjugation

Abstract

Macroautophagy, hereafter autophagy, is a major degradation pathway in eukaryotic systems that allows the removal of large intracellular structures such as entire organelles or protein aggregates, thus contributing to the homeostasis of cells and tissues. Autophagy entails the de novo formation of an organelle termed autophagosome, where a cup-shaped structure called isolation membrane nucleates in proximity of a cytoplasmic cargo material. Upon elongation and closure of isolation membranes, the mature autophagosome delivers the sequestered cargo into the lysosomal system for degradation. Among the factors for autophagosome formation are the autophagy-related (Atg) proteins belonging to the Atg8 conjugation system. In this system, the ubiquitin-like Atg8 protein is conjugated to the membrane lipid phosphatidylethanolamine present in autophagosomal membranes. Atg8 can also be removed from membranes by Atg4-mediated deconjugation. Here, we describe in vitro systems that recapitulate the enzymatic reactions occurring in vivo by presenting expression and purification strategies for all the components of the Saccharomyces cerevisiae Atg8 conjugation system. We also present protocols for in vitro Atg8 conjugation and deconjugation reactions employing small and giant unilamellar vesicles.

Keywords: Atg8 lipidation; Autophagy; GUVs; Isolation membrane; Phosphatidylethanolamine; Reconstitution; SUVs; Ubiquitin-like system.