scholarly journals A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy

2020 ◽  
Vol 219 (7) ◽  
Author(s):  
Dorotea Fracchiolla ◽  
Chunmei Chang ◽  
James H. Hurley ◽  
Sascha Martens
Keyword(s):  
Positive Feedback ◽  
Complex I ◽  
Feedback Loop ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Giant Unilamellar Vesicles ◽  
Autophagosome Formation ◽  
Unilamellar Vesicles ◽  
Positive Feedback Loop ◽  
Double Membrane

Autophagy degrades cytoplasmic cargo by its delivery to lysosomes within double membrane autophagosomes. Synthesis of the phosphoinositide PI(3)P by the autophagic class III phosphatidylinositol-3 kinase complex I (PI3KC3-C1) and conjugation of ATG8/LC3 proteins to phagophore membranes by the ATG12–ATG5-ATG16L1 (E3) complex are two critical steps in autophagosome biogenesis, connected by WIPI2. Here, we present a complete reconstitution of these events. On giant unilamellar vesicles (GUVs), LC3 lipidation is strictly dependent on the recruitment of WIPI2 that in turn depends on PI(3)P. Ectopically targeting E3 to membranes in the absence of WIPI2 is insufficient to support LC3 lipidation, demonstrating that WIPI2 allosterically activates the E3 complex. PI3KC3-C1 and WIPI2 mutually promote the recruitment of each other in a positive feedback loop. When both PI 3-kinase and LC3 lipidation reactions were performed simultaneously, positive feedback between PI3KC3-C1 and WIPI2 led to rapid LC3 lipidation with kinetics similar to that seen in cellular autophagosome formation.

scholarly journals A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy

2019 ◽  
Author(s):  
Dorotea Fracchiolla ◽  
Chunmei Chang ◽  
James H. Hurley ◽  
Sascha Martens
Keyword(s):  
Positive Feedback ◽  
Complex I ◽  
Feedback Loop ◽  
Giant Unilamellar Vesicles ◽  
Autophagosome Formation ◽  
Unilamellar Vesicles ◽  
Positive Feedback Loop ◽  
Double Membrane

AbstractAutophagy degrades cytoplasmic cargo by its delivery to lysosomes within double membrane autophagosomes. Synthesis of the phosphoinositide PI(3)P by the autophagic PI 3-kinase complex I (PI3KC3-C1) and conjugation of ATG8/LC3 proteins to phagophore membranes by the ATG12–ATG5-ATG16L1 (E3) complex are two critical steps in autophagosome biogenesis, connected by WIPI2. Here we present a complete reconstitution of these events. On giant unilamellar vesicles (GUVs), LC3 lipidation is strictly dependent on the recruitment of WIPI2, which in turn depends on PI(3)P. Ectopically targeting E3 to membranes in the absence of WIPI2 is insufficient to support LC3 lipidation, demonstrating that WIPI2 allosterically activates the E3 complex. PI3KC3-C1 and WIPI2 mutually promote the recruitment of each other in a positive feedback loop. When both PI 3-kinase and LC3 lipidation reactions were carried out simultaneously, positive feedback between PI3KC3-C1 and WIPI2 led to rapid LC3 lipidation with kinetics similar to those seen in cellular autophagosome formation.SummaryAutophagy requires the synthesis of PI(3)P and the conjugation of LC3 to the phagophore membrane. We reconstituted these two reactions and their coupling by WIPI2, and showed that positive feedback between PI3KC3-C1 and WIPI2 leads to rapid LC3 lipidation by the ATG16L1 complex.

scholarly journals Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy

2021 ◽  
Vol 7 (17) ◽  
pp. eabg4922
Author(s):  
Chunmei Chang ◽  
Xiaoshan Shi ◽  
Liv E. Jensen ◽  
Adam L. Yokom ◽  
Dorotea Fracchiolla ◽  
...  
Keyword(s):  
Complex I ◽  
Kinase Activity ◽  
Protein Aggregates ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Giant Unilamellar Vesicles ◽  
Core Complex ◽  
Selective Autophagy ◽  
The Core ◽  
Stimulation Of

Selective autophagy of damaged mitochondria, protein aggregates, and other cargoes is essential for health. Cargo initiates phagophore biogenesis, which entails the conjugation of LC3 to phosphatidylethanolamine. Current models suggest that clustered ubiquitin chains on a cargo trigger a cascade from autophagic cargo receptors through the core complexes ULK1 and class III phosphatidylinositol 3-kinase complex I, WIPI2, and the ATG7, ATG3, and ATG12ATG5-ATG16L1 machinery of LC3 lipidation. This was tested using giant unilamellar vesicles (GUVs), GST-Ub4 as a model cargo, the cargo receptors NDP52, TAX1BP1, and OPTN, and the autophagy core complexes. All three cargo receptors potently stimulated LC3 lipidation on GUVs. NDP52- and TAX1BP1-induced LC3 lipidation required all components, but not ULK1 kinase activity. However, OPTN bypassed the ULK1 requirement. Thus, cargo-dependent stimulation of LC3 lipidation is common to multiple autophagic cargo receptors, yet the details of core complex engagement vary between the different receptors.

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