Members of the autophagy class III phosphatidylinositol 3-kinase complex I interact with GABARAP and GABARAPL1 via LIR motifs

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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Class III phosphatidylinositol 3-kinase complex I subunit NRBF2/Atg38 - from cell and structural biology to health and disease

Autophagy ◽

10.1080/15548627.2021.1872240 ◽

2021 ◽

pp. 1-12

Author(s):

Yohei Ohashi

Keyword(s):

Structural Biology ◽

Complex I ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Health And Disease ◽

Phosphatidylinositol 3

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Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

eLife ◽

10.7554/elife.05115 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 79

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.

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STYK1 promotes autophagy through enhancing the assembly of autophagy-specific class III phosphatidylinositol 3-kinase complex I

Autophagy ◽

10.1080/15548627.2019.1687212 ◽

2019 ◽

Vol 16 (10) ◽

pp. 1786-1806 ◽

Cited By ~ 7

Author(s):

Cefan Zhou ◽

Xuehong Qian ◽

Miao Hu ◽

Rui Zhang ◽

Nanxi Liu ◽

...

Keyword(s):

Complex I ◽

Specific Class ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

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Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy

Science Advances ◽

10.1126/sciadv.abg4922 ◽

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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Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression

FEBS Letters ◽

10.1016/j.febslet.2012.04.046 ◽

2012 ◽

Vol 586 (11) ◽

pp. 1584-1591 ◽

Cited By ~ 63

Author(s):

Hilde Abrahamsen ◽

Harald Stenmark ◽

Harald W. Platta

Keyword(s):

Tumor Suppression ◽

Kinase Activity ◽

Beclin 1 ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Binding Partners ◽

Phosphatidylinositol 3

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Class III phosphatidylinositol 3-kinase and its catalytic product PtdIns3P in regulation of endocytic membrane traffic

FEBS Journal ◽

10.1111/febs.12116 ◽

2013 ◽

Vol 280 (12) ◽

pp. 2730-2742 ◽

Cited By ~ 60

Author(s):

Camilla Raiborg ◽

Kay O. Schink ◽

Harald Stenmark

Keyword(s):

Membrane Traffic ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

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Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0080 ◽

2008 ◽

Vol 19 (12) ◽

pp. 5360-5372 ◽

Cited By ~ 715

Author(s):

Eisuke Itakura ◽

Chieko Kishi ◽

Kinji Inoue ◽

Noboru Mizushima

Keyword(s):

Membrane Trafficking ◽

Mammalian Cells ◽

Coiled Coil ◽

Pi3 Kinase ◽

Beclin 1 ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Interacting Protein ◽

Vacuolar Protein ◽

Phosphatidylinositol 3

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.

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Ca2+-regulated Pool of Phosphatidylinositol-3-phosphate Produced by Phosphatidylinositol 3-Kinase C2α on Neurosecretory Vesicles

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0595 ◽

2008 ◽

Vol 19 (12) ◽

pp. 5593-5603 ◽

Cited By ~ 39

Author(s):

Peter J. Wen ◽

Shona L. Osborne ◽

Isabel C. Morrow ◽

Robert G. Parton ◽

Jan Domin ◽

...

Keyword(s):

Critical Role ◽

Neurosecretory Cells ◽

Secretory Vesicles ◽

Endosomal Trafficking ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Product ◽

Phosphatidylinositol 3

Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2α and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2α knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2α knockdown and expression of PI3K-C2α catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca2+. We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2α is indeed regulated by Ca2+. We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2α occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca2+ in vitro and in living neurosecretory cells.

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