Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex

Abstract

Autophagy is an enigmatic cellular process in which double-membrane compartments, called "autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis

“…In these experiments, the membrane curvature of the liposomes did not affect the interaction. In addition, purified ATG2A was shown to bind to liposomes in a PI3P‐independent manner . In contrast to ATG2B, however, the size of liposomes markedly affected their affinity to ATG2A.…”

“…Although structural studies on the core Atg proteins have significantly progressed in the last 15 years, the structure of Atg2 remained unsolved until 2017 due to its large size, 180 kDa in budding yeast, and the tendency of proteins to aggregate, making crystallization and NMR analysis difficult. However, recent advances in electron microscopy (EM) enabled the imaging of the mammalian Atg2–Atg18 complex at a low resolution . Mammals have two Atg2 homologs (ATG2A and 2B) and four Atg18 homologs, known as WD repeat domain phosphoinositide‐interacting proteins (WIPI) 1–4 .…”

“…Negative‐staining EM analysis of the ATG2A–WIPI4 complex (WIPI4 is also known as WDR45) and the ATG2B–WIPI4 complex revealed the overall shape of this protein complex (Fig. , left) . ATG2 has a rod‐like structure with a length of approximately 20 nm.…”

“…The β‐propeller ring of WIPI4 binds to one end of the ATG2 rod. Thus, the overall shape of the ATG2–WIPI4 complex looks like a golf club . This architecture is roughly similar to a multiple‐subunit tethering complex, such as homotypic fusion and vacuole protein sorting, but the lack of high‐resolution structural information for Atg2 hampers further elucidation of the molecular functions of this complex family during autophagosome formation.…”

“…Structure of Atg2. Left, shape of the ATG2A–WIPI4 complex revealed by negative‐staining EM (EMDB ID 8899) . Ribbon model represents the crystal structure of WIPI3 (PDB ID 6IYY) manually fitted into the EM image.…”