Autophagosome formation is initiated at phosphatidylinositol synthase‐enriched ER subdomains

Abstract

The autophagosome, a double-membrane structure mediating degradation of cytoplasmic materials by macroautophagy, is formed in close proximity to the endoplasmic reticulum (ER). However, how the ER membrane is involved in autophagy initiation and to which membrane structures the autophagy-initiation complex is localized have not been fully characterized. Here, we were able to biochemically analyze autophagic intermediate membranes and show that the autophagy-initiation complex containing ULK and FIP200 first associates with the ER membrane. To further characterize the ER subdomain, we screened phospholipid biosynthetic enzymes and found that the autophagy-initiation complex localizes to phosphatidylinositol synthase (PIS)-enriched ER subdomains. Then, the initiation complex translocates to the ATG9A-positive autophagosome precursors in a PI3P-dependent manner. Depletion of phosphatidylinositol (PI) by targeting bacterial PI-specific phospholipase C to the PIS domain impairs recruitment of downstream autophagy factors and autophagosome formation. These findings suggest that the autophagy-initiation complex, the PIS-enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.

Targeting autophagy in cardiac ischemia/reperfusion injury: A novel therapeutic strategy

“…PI(3)P recruits the PI (3) P-binding proteins DFCP1 (double FYVE-domain-containing protein 1) and WIPI-1/2 (WD repeat domain phosphoinositide-interacting proteins 1/2) to the omegasome, further leading to growth and expansion of the omegasome (Ravikumar et al, 2010). In the expansion step, Atg9-containing vesicles also may deliver other components, such as proteins and lipids to contribute to omegasome growth (Karanasios et al, 2013;Manifava et al, 2016;Nishimura et al, 2017). The most important step of omegasome expansion is mediated by the Atg conjugation system, which requires an ubiquitin-like system (Nakatogawa, 2013).…”

The where, what, and when of membrane protein degradation in neurons

“…In (macro-) autophagy, a double membrane called phagophore forms around "to-be-degraded" cargo, such as vesicles containing membrane proteins, cytosolic proteins, protein aggregates, and organelles. After cargo engulfment, autophagosomes fuse with lysosomal vesicles to form degradative autolysosomes (Xie and Klionsky, 2007;Kraft and Martens, 2012;Coutts and La Thangue, 2016;Nishimura et al, 2017;Wang et al, 2017). Despite the abundance of transmembrane proteins in both presynaptic and postsynaptic compartments, progress has only recently been made to address what membrane proteins are degraded by either mechanism in neurons (Ashrafi and Schwarz, 2013;Huber and Teis, 2016;Mancias and Kimmelman, 2016;Zaffagnini and Martens, 2016;Vijayan and Verstreken, 2017).…”

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution

“…Interestingly, PIS1 subdomains have been proposed to be involved in the delivery of PtdIns to a variety of cellular membranes, including endosomes . Moreover, a recent paper from Mizushima's group demonstrated that autophagosome formation occurs preferentially in enriched PIS1 subdomains in the ER . As VMP1 has been implicated in both ER‐endosome contact sites and autophagosome biogenesis, we analyzed VMP1 and PIS1 subdomains with respect to endosomes and autophagosomes.…”

“…33 Moreover, a recent paper from Mizushima's group demonstrated that autophagosome formation occurs preferentially in enriched PIS1 subdomains in the ER. 34 As VMP1 has been implicated in both ER-endosome contact sites 18 and autophagosome biogenesis, 12,14 we analyzed VMP1 and PIS1 subdomains with respect to endosomes and autophagosomes. We found that most of the VMP1-PIS1 positive subdomains colocalize with LC3-labeled autophagosomes and EGFRlabeled endosomes, although in different proportions ( Figure 4).…”