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.

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.

The class III phosphatidylinositol 3-kinase Vps34 in Saccharomyces cerevisiae

2017 ◽  
Vol 398 (5-6) ◽  
pp. 677-685 ◽  
Author(s):  
Christina Reidick ◽  
Fahd Boutouja ◽  
Harald W. Platta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Role ◽  
Protein Sorting ◽  
Endocytic Trafficking ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

Abstract The class III phosphatidylinositol 3-kinase Vps34 (vacuolar protein sorting 34) catalyzes for the formation of the signaling lipid phosphatidylinositol-3-phopsphate, which is a central factor in the regulation of autophagy, endocytic trafficking and vesicular transport. In this article, we discuss the functional role of the lipid kinase Vps34 in Saccharomyces cerevisiae.

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

2008 ◽  
Vol 19 (12) ◽  
pp. 5360-5372 ◽  
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.

scholarly journals Assortment of Phosphatidylinositol 3-Kinase Complexes—Atg14p Directs Association of Complex I to the Pre-autophagosomal Structure in Saccharomyces cerevisiae

2006 ◽  
Vol 17 (4) ◽  
pp. 1527-1539 ◽  
Author(s):  
Keisuke Obara ◽  
Takayuki Sekito ◽  
Yoshinori Ohsumi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Complex I ◽  
Regulatory Subunit ◽  
Biological Processes ◽  
Phosphatidylinositol 3 Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Complex Ii ◽  
Vacuolar Protein ◽  
Vacuolar Membranes ◽  
Phosphatidylinositol 3

In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.

scholarly journals Structural Pathway for Allosteric Activation of the Autophagic PI 3-Kinase Complex I

2019 ◽  
Author(s):  
Lindsey N. Young ◽  
Felix Goerdeler ◽  
James H. Hurley
Keyword(s):  
Protein Kinase ◽  
Complex I ◽  
Structural Changes ◽  
Kinase Domain ◽  
Activation Loop ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Autophagy Induction ◽  
Enzymatic Activation

AbstractAutophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the pro-autophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryo-EM was used to visualize stepwise PI3KC3-C1 activating effects of binding the NRFB2 MIT domains. Binding of a single NRBF2 MIT domain to bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.

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

Autophagy ◽  
2019 ◽  
Vol 16 (10) ◽  
pp. 1786-1806 ◽  
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

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

Autophagy ◽  
2019 ◽  
Vol 15 (8) ◽  
pp. 1333-1355 ◽  
Author(s):  
Åsa Birna Birgisdottir ◽  
Stephane Mouilleron ◽  
Zambarlal Bhujabal ◽  
Martina Wirth ◽  
Eva Sjøttem ◽  
...  
Keyword(s):  
Complex I ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

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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