Authors:	Schmitt, Daniel Bozkurt, Süleyman Henning-Domres, Pascale Huesmann, Heike Eimer, Stefan Bindila, Laura Behrends, Christian Boyle, Emily Wilfling, Florian Tascher, Georg Münch, Christian Behl, Christian Kern, Andreas
Title:	Lipid and protein content profiling of isolated native autophagic vesicles
Online publication date:	10-Jan-2023
Year of first publication:	2022
Language:	english
Abstract:	Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin–proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses.
DDC:	610 Medizin 610 Medical sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 04 Medizin
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-8543
Version:	Published version
Publication type:	Zeitschriftenaufsatz
License:	CC BY-NC-ND
Information on rights of use:	https://creativecommons.org/licenses/by-nc-nd/4.0/
Journal:	EMBO reports 23
Pages or article number:	e53065
Publisher:	EMBO Press
Publisher place:	Heidelberg
Issue date:	2022
ISSN:	1469-3178
Publisher DOI:	10.15252/embr.202153065
Appears in collections:	DFG-491381577-H