Abstract
Autophagy is a cytosolic quality control process that recognizes substrates through receptor‐mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR‐Cas9 or knockout‐mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER‐resident lectin chaperone Calnexin (CANX) and the ER‐phagy receptor FAM134B are required for autophagy‐mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co‐receptor that recognizes ER luminal misfolded procollagens and interacts with the ER‐phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane‐associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome‐resistant misfolded clients from the ER.
Highlights
Macroautophagy is a homeostatic catabolic process devoted to the sequestration of cytoplasmic material in double-membrane vesicles that eventually fuse with lysosomes where cargo is degraded (Mizushima, 2011)
Using three different collagen producing cell lines, mouse embryonic fibroblasts (MEFs) and human osteoblasts (Saos2) stably expressing the autophagosome membrane marker LC3 fused with GFP (GFPLC3) (Kabeya et al, 2000), and rat chondrosarcoma cells (RCS) immunolabelled for LC3, we observed co-localization of LC3-positive vesicles with PC1 (MEFs and Saos2) and PC2 (RCS) (Fig 1A–D)
When MEFs, Saos2 and RCS cells were treated with the lysosomal inhibitor bafilomycin A1 (BafA1), PC molecules accumulated in the lumen of swollen endo/lysosomes (LAMP1-positive organelles, hereafter referred as lysosomes) (Fig 1E–G)
Summary
Macroautophagy (hereafter referred to as autophagy) is a homeostatic catabolic process devoted to the sequestration of cytoplasmic material in double-membrane vesicles (autophagic vesicles, AVs) that eventually fuse with lysosomes where cargo is degraded (Mizushima, 2011). Autophagy receptors harbour a LC3 or GABARAP interaction motif (LIR or GIM, respectively) that facilitate binding of the cargo to LC3 or GABARAP proteins, which decorate autophagosomal membranes (Stolz et al, 2014; Rogov et al, 2017). Proteins and entire organelles or their portions can be targeted to autophagy via receptor-mediated processes. A notable example is represented by ERphagy, a selective form of autophagy in which portions of the ER are sequestered within AVs and transported to the lysosomes for degradation (Fregno & Molinari, 2018; Grumati et al, 2018).
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