Characterization of Autophagy Cargo Profiles during Starvation and Exercise with a Novel Autophagosome Isolation Method

Abstract

Autophagy is a cellular process to clear unwanted and dysfunctional cellular cargoes, which are sequestered in autophagosomes before their delivery to lysosomes for degradation. Autophagy cargo selection, mediated by cargo receptors, varies across cell types and conditions. Understanding the cargo features is essential for elucidating autophagys function in specific physiological or pathological contexts. Here we present a simple and rapid method for isolating LC3B-positive autophagosomes from the tissues of GFP-LC3 transgenic mice, a widely used autophagy reporter model, without relying on the complex ultracentrifugation steps required by traditional methods. When combined with quantitative proteomics, this approach enables efficient in vivo characterization of autophagy cargoes. We applied this method to establish autophagy cargo profiles in skeletal muscle during starvation and exercise, two physiological conditions that activate autophagy, and identified distinct cargo selection patterns, with significantly higher levels of ER-phagy and ribophagy observed during starvation. We further revealed the ER-phagy receptors TEX264 and RETREG1/FAM134B as potential mediators of the elevated ER-phagy under starvation. In summary, we report an efficient workflow for in vivo autophagy cargo characterization and provide detailed analysis and comparison of cargo profiles under starvation and exercise conditions.