Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

Ohashi, Yohei; Soler, Nicolas; Ortegón, Miguel García; Zhang, Lufei; Kirsten, Marie L.; Perišić, Olga; Masson, Glenn R.; Burke, John E.; Jakobi, Arjen J.; Apostolakis, Apostolos A.; Johnson, Christopher M.; Ohashi, Maki; Ktistakis, Nicholas T.; Sachse, Carsten; Williams, Roger L.

doi:10.1080/15548627.2016.1226736

Cited by 56 publications

(70 citation statements)

References 32 publications

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“…PI3KC3-C1 associates tightly with a fifth subunit, known as NRBF2 (Nuclear Receptor Binding Factor 2) (101–103) in mammals and Atg38 (104) in yeast. NRBF2/Atg38 contain an N-terminal three-helix bundle MIT (Microtubule-Interacting and Transport) domain and a coiled-coil-containing domain at the C-terminus (105) that induces dimerization. NRBF2 binds to the base of complex I through interactions with the N-terminus of ATG14L and BECN1, enhances kinase activity in vitro and leads to the dimerization of human complex I (106) (Fig 3B).…”

Section: Pi3kc3-c1 Regulatory Proteinsmentioning

confidence: 99%

“…NRBF2 binds to the base of complex I through interactions with the N-terminus of ATG14L and BECN1, enhances kinase activity in vitro and leads to the dimerization of human complex I (106) (Fig 3B). Although yeast Atg38 is dimeric as well, it does not facilitate yeast PI3KC3-C1 dimerization (105). Mammalian PI3KC3-C1 dimerization and kinase activation by NRBF2 seem to be completely decoupled from one another (106).…”

Section: Pi3kc3-c1 Regulatory Proteinsmentioning

confidence: 99%

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Mechanisms of Autophagy Initiation

Hurley

Young

2017

Annu. Rev. Biochem.

544

424

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Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 protein kinase complex and the PI3KC3-C1 lipid kinase complex. Some 90 % of the mass of these large protein complexes consists on non-catalytic domains and subunits, and the ULK1 complex has essential non-catalytic activities. Structural studies of these complexes have shed increasing light on the regulation of their catalytic and non-catalytic activities in autophagy initiation. The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9, COPII vesicles, and possibly other sources. In the wake of reconstitution and superresolution imaging studies, we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.

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Section: Pi3kc3-c1 Regulatory Proteinsmentioning

confidence: 99%

Section: Pi3kc3-c1 Regulatory Proteinsmentioning

confidence: 99%

Mechanisms of Autophagy Initiation

Hurley

Young

2017

Annu. Rev. Biochem.

544

424

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“…The N-terminal regulatory sequences of ATG14 and BECN1 have not been visualized in either the EM or crystal structures, but are presumably attached to the base. These regions contain the binding sites for important regulators of PI3KC3 such as Bcl-2 (Oberstein et al, 2007; Pattingre et al, 2005) and NRBF2 (Ohashi et al, 2016; Young et al, 2016) and important sites of regulatory phosphorylation by ULK1, AMPK, DAPK, MAPKAP2, and PGK1 (Kim et al, 2013; Qian et al, 2017; Russell et al, 2013; Wei et al, 2015). The VPS34 catalytic subunit makes almost no contacts between the regulatory BECN1 and ATG14 subunits.…”

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confidence: 99%

Vps34 Kinase Domain Dynamics Regulate the Autophagic PI 3-Kinase Complex

et al. 2017

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Summary The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is required for the initiation of essentially all macroautophagic processes. PI3KC3-C1 consists of the lipid kinase catalytic subunit VPS34, the VPS15 scaffold, and the regulatory BECN1 and ATG14 subunits. The VPS34 catalytic domain and BECN1:ATG14 subcomplex do not touch, and it is unclear how allosteric signals are transmitted to VPS34. We used EM and cross-linking mass spectrometry to dissect five conformational substates of the complex, including one in which the VPS34 catalytic domain is dislodged from the complex but remains tethered by an intrinsically disordered linker. A “leashed” construct prevented dislodging without interfering with the other conformations, blocked enzyme activity in vitro, and blocked autophagy induction in yeast cells. This pinpoints the dislodging and tethering of the VPS34 catalytic domain, and its regulation by VPS15, as a master allosteric switch in autophagy induction.

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“…The pro-autophagic function of NRBF2 has been confirmed by most reports in mammalian cells (20)(21)(22)(23)(24), although contrary findings were reported by one group (24). NRBF2 and its ortholog Atg38 contain an N-terminal Microtubule Interacting and Trafficking (MIT) domain that binds to PI3KC3-C1, followed by a central dimeric coiledcoil domain (17,19,25) (Fig. 1B).…”

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confidence: 52%

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

Young

Goerdeler

Hurley

2019

Preprint

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Autophagy 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.Autophagy is a core cellular process, conserved throughout eukaryotes, which is the central recycling system for the removal of misfolded proteins, damaged organelles, and the recycling of nutrients in starvation. Autophagic dysfunction is implicated in many disease states, including neurodegeneration, immune disorders, cancer, and aging, among others (1). The class III phosphatidylinositol-3 kinase complexes (PI3KC3) I and II (PI3KC3-C1 and -C2), respectively, are essential for the initiation and expansion of autophagosomes (2-5). PI3KC3 generates the lipid phosphatidylinositol-3-phosphate, PI(3)P, which is recognized by the WIPI proteins. WIPIs in turn recruit the machinery that conjugates the autophagosomal marker LC3 to the expanding autophagosomal membrane (6). PI3KC3-C1 has been proposed to be a promising therapeutic target for autophagy activators (7) because the generation of PI(3)P is absolutely required for the recruitment of downstream autophagy proteins. There is considerable medical interest in selectively activating this pathway to promote human health and treat disease, yet there are no FDA approved pharmaceuticals that uniquely activate autophagy.PI3KC3-C1 consists of the lipid kinase VPS34, the putative serine/threonine protein kinase VPS15, the regulatory subunit BECN1, and the early autophagy-specific targeting subunit ATG14 (8,9). In PI3KC3-C2, ATG14 is replaced with UVRAG (10), while the other three subunits are preserved. The overall architecture of both PI3KC3-C1 and -C2 has the shape of the letter V (11, 12) (Fig. 1A). The long coiled-coils of BECN1 and ATG14 scaffold the left arm in the standard view, with the membrane binding BARA domain of BECN1 located at the outermost tip of the arm. PI3KC3-C2 is inhibited by Rubicon and the HIV-1 protein Nef (13-15), which regulate membrane docking by the tip of the left arm. The catalytic domains of the kinases VPS34 and VPS15 are at the tip of the right arm (11,12). PI3KC3 complexes are phosphoregulated by the Unc-51 like autophagy-activating kinase 1 (ULK1) complex (16),

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Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

Cited by 56 publications

References 32 publications

Mechanisms of Autophagy Initiation

Mechanisms of Autophagy Initiation

Vps34 Kinase Domain Dynamics Regulate the Autophagic PI 3-Kinase Complex

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

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