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

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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Possible involvement of Class III phosphatidylinositol-3-kinase in meiotic progression of porcine oocytes beyond germinal vesicle stage

Theriogenology ◽

10.1016/j.theriogenology.2010.11.002 ◽

2011 ◽

Vol 75 (5) ◽

pp. 940-950 ◽

Cited By ~ 4

Author(s):

Myung Rae Park ◽

Mukesh Kumar Gupta ◽

Hye Ran Lee ◽

Ziban Chandra Das ◽

Sang Jun Uhm ◽

...

Keyword(s):

Germinal Vesicle ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Meiotic Progression ◽

Phosphatidylinositol 3

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603650113 ◽

2016 ◽

Vol 113 (29) ◽

pp. 8224-8229 ◽

Cited By ~ 34

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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Phosphatidylinositol 3,5-bisphosphate plays a role in the activation and subcellular localization of mechanistic target of rapamycin 1

Molecular Biology of the Cell ◽

10.1091/mbc.e11-12-1034 ◽

2012 ◽

Vol 23 (15) ◽

pp. 2955-2962 ◽

Cited By ~ 81

Author(s):

Dave Bridges ◽

Jing-Tyan Ma ◽

Sujin Park ◽

Ken Inoki ◽

Lois S. Weisman ◽

...

Keyword(s):

Plasma Membrane ◽

Subcellular Localization ◽

Target Of Rapamycin ◽

Class Iii ◽

Cell Type ◽

Phosphatidylinositol 3 Kinase ◽

Mechanistic Target Of Rapamycin ◽

Cell Type Specific ◽

Stepwise Formation ◽

Phosphatidylinositol 3

The kinase complex mechanistic target of rapamycin 1 (mTORC1) plays an important role in controlling growth and metabolism. We report here that the stepwise formation of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) regulates the cell type–specific activation and localization of mTORC1. PI(3)P formation depends on the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α, as well as the class III PI3K Vps34, while PI(3,5)P2 requires the phosphatidylinositol-3-phosphate-5-kinase PIKFYVE. In this paper, we show that PIKFYVE and PI3K-C2α are necessary for activation of mTORC1 and its translocation to the plasma membrane in 3T3-L1 adipocytes. Furthermore, the mTORC1 component Raptor directly interacts with PI(3,5)P2. Together these results suggest that PI(3,5)P2 is an essential mTORC1 regulator that defines the localization of the complex.

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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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Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation

The Journal of Cell Biology ◽

10.1083/jcb.200107069 ◽

2001 ◽

Vol 155 (1) ◽

pp. 19-26 ◽

Cited By ~ 346

Author(s):

Otilia V. Vieira ◽

Roberto J. Botelho ◽

Lucia Rameh ◽

Saskia M. Brachmann ◽

Tsuyoshi Matsuo ◽

...

Keyword(s):

Functional Role ◽

Fluorescent Proteins ◽

Class I ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Intracellular Parasites ◽

Selective Ablation ◽

Phagosomal Membrane ◽

Phosphatidylinositol 3

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3′-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

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