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.

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

2021 ◽  
Author(s):  
Chunmei Chang ◽  
Xiaoshan Shi ◽  
Liv Jensen ◽  
Adam L Yokom ◽  
Dorotea Fracchiolla ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Complex I ◽  
Common Property ◽  
Kinase Activity ◽  
Intracellular Pathogens ◽  
Class Iii ◽  
Giant Unilamellar Vesicles ◽  
Core Complex ◽  
Selective Autophagy ◽  
Stimulation Of

Selective autophagy of damaged mitochondria, intracellular pathogens, protein aggregates, endoplasmic reticulum, and other large cargoes is essential for health. The presence of cargo initiates phagophore biogenesis, which entails the conjugation of ATG8/LC3 family proteins to membrane phosphatidylethanolamine. Current models suggest that the presence of clustered ubiquitin chains on a cargo triggers a cascade of interactions from autophagic cargo receptors through the autophagy core complexes ULK1 and class III PI 3-kinase complex I (PI3KC3-C1), WIPI2, and the ATG7, ATG3, and ATG12-ATG5-ATG16L1 machinery of LC3 lipidation. This model 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 the ULK1 complex together with all other components, however, ULK1 kinase activity was dispensable. In contrast, OPTN bypassed the ULK1 requirement completely. These data show that the cargo-dependent stimulation of LC3 lipidation is a common property of multiple autophagic cargo receptors, yet the details of core complex engagement vary considerably and unexpectedly between the different receptors.

scholarly journals Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Sulochanadevi Baskaran ◽  
Lars-Anders Carlson ◽  
Goran Stjepanovic ◽  
Lindsey N Young ◽  
Do Jin Kim ◽  
...  
Keyword(s):  
Complex I ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Scaffolding Protein ◽  
Hydrogen Deuterium Exchange ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Hydrogen Deuterium ◽  
Phosphatidylinositol 3

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

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 Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

2016 ◽  
Vol 113 (29) ◽  
pp. 8224-8229 ◽  
Author(s):  
Lindsey N. Young ◽  
Kelvin Cho ◽  
Rosalie Lawrence ◽  
Roberto Zoncu ◽  
James H. Hurley
Keyword(s):  
Complex I ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Negative Stain ◽  
Binding Factor ◽  
Hydrogen Deuterium ◽  
Negative Stain Electron Microscopy ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen–deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure.

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