Abstract
Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes. DOI:http://dx.doi.org/10.7554/eLife.00947.001.
Highlights
Autophagy is a conserved catabolic process underlying the self-digestion of cytoplasmic components through the formation of double-membraned vesicles termed autophagosomes
A key step in autophagosome biogenesis is the generation of PE-lipidated LC3 by a ubiquitin-like conjugation system (Ichimura et al, 2000; Kabeya et al, 2000)
In vitro LC3 lipidation has recently been reconstituted with synthetic liposomes, recombinant LC3 and other components including the ATG12–ATG5 conjugate, ATG7 and ATG3 (Sou et al, 2006; Hanada et al, 2007; Oh-oka et al, 2008; Shao et al, 2007; Gao et al, 2010)
Summary
Autophagy is a conserved catabolic process underlying the self-digestion of cytoplasmic components through the formation of double-membraned vesicles termed autophagosomes. In the process of starvation-induced autophagy, several upstream signals are triggered, including inhibition of the mechanistic target of rapamycin (MTOR), and activation of the Jun N-terminal kinase (JNK) and AMP kinase (AMPK) (Noda and Ohsumi, 1998; Wei et al, 2008; Kim et al, 2011; Rubinsztein et al, 2012; Kim et al, 2013) These signals are conveyed to activate the serine/threonine-protein kinase complex containing the Atg homologs ULK1/2, ATG13, FIP200 (RB1CC1) and ATG101 (C12orf44) (Mizushima, 2010). DFCP1 (ZFYVE1), an endoplasmic reticulum (ER)-associated PI3P binding protein, is recruited to the site of newly-generated PI3P to form omegasomes (Axe et al, 2008) This is followed by two ubiquitin-like conjugation systems to produce the ATG12–ATG5 conjugate and phosphatidylethanolamine (PE)lipidated ATG8/LC3, which initiates the formation of a preautophagosomal organelle termed the phagophore or isolation membrane (Mizushima et al, 1998a, 1998b; Ichimura et al, 2000; Geng and Klionsky, 2008). The crescent-shaped tubular membrane seals to form a double-membraned autophagosome with cytoplasmic
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