Local Fatty Acid Channeling into Phospholipid Synthesis Drives Phagophore Expansion during Autophagy.

Autophagy is a conserved catabolic homeostasis process central for cellular and organismal health. During autophagy, small single-membrane phagophores rapidly expand into large double-membrane autophagosomes to encapsulate diverse cargoes for degradation. It is thought that autophagic membranes are mainly derived from preformed organelle membranes. Instead, here we delineate a pathway that expands the phagophore membrane by localized phospholipid synthesis. Specifically, we find that the conserved acyl-CoA synthetase Faa1 accumulates on nucleated phagophores and locally activates fatty acids (FAs) required for phagophore elongation and autophagy. Strikingly, using isotopic FA tracing, we directly show that Faa1 channels activated FAs into the synthesis of phospholipids and promotes their assembly into autophagic membranes. Indeed, the first committed steps of de novo phospholipid synthesis at the ER, which forms stable contacts with nascent autophagosomes, are essential for autophagy. Together, our work illuminates how cells spatially tune synthesis and flux of phospholipids for autophagosome biogenesis during autophagy.

Emerging roles of ATG9/ATG9A in autophagy: implications for cell and neurobiology.

Atg9, the only transmembrane protein among many autophagy-related proteins, was first identified in the year 2000 in yeast. Two homologs of Atg9, ATG9A and ATG9B, have been found in mammals. While ATG9B shows a tissue-specific expression pattern, such as in the placenta and pituitary gland, ATG9A is ubiquitously expressed. Additionally, ATG9A deficiency leads to severe defects not only at the molecular and cellular levels but also at the organismal level, suggesting key and fundamental roles for ATG9A. The subcellular localization of ATG9A on small vesicles and its functional relevance to autophagy have suggested a potential role for ATG9A in the lipid supply during autophagosome biogenesis. Nevertheless, the precise role of ATG9A in the autophagic process has remained a long-standing mystery, especially in neurons. Recent findings, however, including structural, proteomic, and biochemical analyses, have provided new insights into its function in the expansion of the phagophore membrane. In this review, we aim to understand various aspects of ATG9 (in invertebrates and plants)/ATG9A (in mammals), including its localization, trafficking, and other functions, in nonneuronal cells and neurons by comparing recent discoveries related to ATG9/ATG9A and proposing directions for future research.Abbreviation: AP-4: adaptor protein complex 4; ATG: autophagy related; cKO: conditional knockout; CLA-1: CLArinet (functional homolog of cytomatrix at the active zone proteins piccolo and fife); cryo-EM: cryogenic electron microscopy; ER: endoplasmic reticulum; KO: knockout; PAS: phagophore assembly site; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SV: synaptic vesicle; TGN: trans-Golgi network; ULK: unc-51 like autophagy activating kinase; WIPI2: WD repeat domain, phosphoinositide interacting 2.

The autophagic membrane tether ATG2A transfers lipids between membranes.

An enigmatic step in de novo formation of the autophagosome membrane compartment is the expansion of the precursor membrane phagophore, which requires the acquisition of lipids to serve as building blocks. Autophagy-related 2 (ATG2), the rod-shaped protein that tethers phosphatidylinositol 3-phosphate (PI3P)-enriched phagophores to the endoplasmic reticulum (ER), is suggested to be essential for phagophore expansion, but the underlying mechanism remains unclear. Here, we demonstrate that human ATG2A is a lipid transfer protein. ATG2A can extract lipids from membrane vesicles and unload them to other vesicles. Lipid transfer by ATG2A is more efficient between tethered vesicles than between untethered vesicles. The PI3P effectors WIPI4 and WIPI1 associate ATG2A stably to PI3P-containing vesicles, thereby facilitating ATG2A-mediated tethering and lipid transfer between PI3P-containing vesicles and PI3P-free vesicles. Based on these results, we propose that ATG2-mediated transfer of lipids from the ER to the phagophore enables phagophore expansion.

TOM40 Targets Atg2 to Mitochondria-Associated ER Membranes for Phagophore Expansion.

During autophagy, phagophores grow into double-membrane vesicles called autophagosomes, but the underlying mechanism remains unclear. Here, we show a critical role of Atg2A in phagophore expansion. Atg2A translocates to the phagophore at the mitochondria-associated ER membrane (MAM) through a C-terminal 45-amino acid domain that we have termed the MAM localization domain (MLD). Proteomic analysis identifies the outer mitochondrial membrane protein TOM40 as a MLD-interacting partner. The Atg2A-TOM40 interaction is responsible for MAM localization of Atg2A and requires the TOM receptor protein TOM70. In addition, Atg2A interacts with Atg9A by a region within its N terminus. Inhibition of either Atg2A-TOM40 or Atg2A-Atg9A interactions impairs phagophore expansion and accumulates Atg9A-vesicles in the vicinity of autophagic structures. Collectively, we propose a model that the TOM70-TOM40 complex recruits Atg2A to the MAM for vesicular and/or non-vesicular lipid transport into the expanding phagophore to grow the size of autophagosomes for efficient autophagic flux.

ATG2 regulation of phagophore expansion at mitochondria-associated ER membranes.

The mechanism by which ATG2 (ATG2A and ATG2B in mammals) regulates autophagosome biogenesis remains largely unknown. In our recent study, we showed that ATG2A translocates to the mitochondria-associated ER membranes (MAM) to promote phagophore growth during nutrient starvation. Mechanistically, the mitochondrial translocase TOMM40 binds to a C-terminal domain of ATG2A, termed the MAM localization domain (MLD), and mediates its MAM translocation in a manner dependent on the TOMM receptor TOMM70. Moreover, ATG2A associates with ATG9A through its N-terminal domain and this interaction is required for phagophore expansion and efficient autophagic flux. These observations suggest that ATG2 operates a mechanism for phagophore expansion at the MAM through the TOMM40-TOMM70 complex and ATG9 during autophagy.