title: Nrbf2 protein suppresses autophagy by modulating Atg14L protein-containing Beclin 1-Vps34 complex architecture and reducing intracellular phosphatidylinositol-3 phosphate levels.
abstract: Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1(-/-);Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhong
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Deanna H
authors.initials: DH
authors.lastname: Morris
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Lin
authors.initials: L
authors.lastname: Jin
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Mittul S
authors.initials: MS
authors.lastname: Patel
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Senthil K
authors.initials: SK
authors.lastname: Karunakaran
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: You-Jun
authors.initials: YJ
authors.lastname: Fu
authors.affiliation: Graduate Center for Toxicology, and University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Matuszak
authors.affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536 and.
authors.forename: Heidi L
authors.initials: HL
authors.lastname: Weiss
authors.affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, New York, 10065.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536; Graduate Center for Toxicology, and University of Kentucky, Lexington, Kentucky 40536; Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536 and. Electronic address: qingjun.wang@uky.edu.
authors.forename: Qing Jun
authors.initials: QJ
authors.lastname: Wang
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D001343:Autophagy; D000071183:Autophagy-Related Proteins; D000071186:Beclin-1; D058546:Class III Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D056945:Hep G2 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D060066:Protein Interaction Maps; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology*
chemical_list: C539000:ATG14 protein, human; D033942:Adaptor Proteins, Vesicular Transport; D051017:Apoptosis Regulatory Proteins; D000071183:Autophagy-Related Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C494986:NRBF2 protein, human; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C485184:enhanced green fluorescent protein; C055525:phosphatidylinositol 3-phosphate; D049452:Green Fluorescent Proteins; D058546:Class III Phosphatidylinositol 3-Kinases
keywords: Autophagosome Biogenesis; Autophagy; Beclin 1; Cellular Regulation; Nrbf2; Protein Degradation; Protein-Protein Interaction; Subcellular Organelle; Vps34
references.citation: J Biol Chem. 2009 May 1;284(18):12297-305
references.pmid: 19258318
references.citation: Neuron. 2002 Aug 29;35(5):921-33
references.pmid: 12372286
references.citation: Autophagy. 2008 Oct;4(7):849-50
references.pmid: 18758232
references.citation: Cell Death Differ. 2011 Oct;18(10):1598-607
references.pmid: 21475306
references.citation: Nat Commun. 2012 Feb 07;3:662
references.pmid: 22314358
references.citation: Nature. 2008 Feb 28;451(7182):1069-75
references.pmid: 18305538
references.citation: Proc Natl Acad Sci U S A. 2005 Oct 4;102(40):14238-43
references.pmid: 16176982
references.citation: J Cell Sci. 2013 Nov 15;126(Pt 22):5224-38
references.pmid: 24013547
references.citation: Cell. 2005 Sep 23;122(6):927-39
references.pmid: 16179260
references.citation: Genomics. 1999 Jul 1;59(1):59-65
references.pmid: 10395800
references.citation: Science. 2012 Nov 16;338(6109):956-9
references.pmid: 23112296
references.citation: Nature. 2007 Jun 28;447(7148):1121-5
references.pmid: 17589504
references.citation: Curr Opin Cell Biol. 2010 Apr;22(2):140-9
references.pmid: 20097051
references.citation: Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19211-6
references.pmid: 19050071
references.citation: J Biol Chem. 1994 Dec 16;269(50):31552-62
references.pmid: 7989323
references.citation: EMBO J. 1995 Jul 17;14(14):3339-48
references.pmid: 7628435
references.citation: Science. 2003 Sep 5;301(5638):1387-91
references.pmid: 12958363
references.citation: Autophagy. 2010 Aug;6(6):764-76
references.pmid: 20639694
references.citation: J Virol. 1998 Nov;72(11):8586-96
references.pmid: 9765397
references.citation: J Biol Chem. 2006 May 19;281(20):14474-85
references.pmid: 16522639
references.citation: J Invest Dermatol. 2004 Dec;123(6):1092-101
references.pmid: 15610520
references.citation: J Clin Invest. 2003 Dec;112(12):1809-20
references.pmid: 14638851
references.citation: Autophagy. 2009 Jul;5(5):720-2
references.pmid: 19395874
references.citation: Nat Commun. 2014 May 22;5:3920
references.pmid: 24849286
references.citation: Sci Rep. 2013;3:1055
references.pmid: 23316280
references.citation: J Biol Chem. 2007 Dec 21;282(51):37124-33
references.pmid: 17940279
references.citation: J Cell Biol. 2010 Aug 23;190(4):511-21
references.pmid: 20713597
references.citation: J Cell Biol. 2001 Feb 5;152(3):519-30
references.pmid: 11157979
references.citation: J Cell Biol. 2008 Aug 25;182(4):685-701
references.pmid: 18725538
references.citation: EMBO J. 2007 May 16;26(10):2527-39
references.pmid: 17446862
references.citation: Nature. 2010 Jul 1;466(7302):68-76
references.pmid: 20562859
references.citation: Cell. 2010 Feb 5;140(3):313-26
references.pmid: 20144757
references.citation: Cell Death Differ. 2011 Apr;18(4):571-80
references.pmid: 21311563
references.citation: Nature. 2013 Mar 21;495(7441):389-93
references.pmid: 23455425
references.citation: Nat Cell Biol. 2006 Jul;8(7):688-99
references.pmid: 16799551
references.citation: J Cell Biol. 2005 Nov 21;171(4):603-14
references.pmid: 16286508
references.citation: Trends Cell Biol. 2010 Jun;20(6):355-62
references.pmid: 20356743
references.citation: J Cell Biol. 2008 May 5;181(3):497-510
references.pmid: 18443221
references.citation: EMBO J. 2000 Sep 1;19(17):4577-88
references.pmid: 10970851
references.citation: Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):20364-71
references.pmid: 24277826
references.citation: Nat Cell Biol. 2009 Apr;11(4):468-76
references.pmid: 19270693
references.citation: Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):15077-82
references.pmid: 14657337
references.citation: FEBS Lett. 2007 May 22;581(11):2156-61
references.pmid: 17382324
references.citation: J Neurotrauma. 2005 Jul;22(7):750-62
references.pmid: 16004578
references.citation: Mol Biol Cell. 2008 Dec;19(12):5360-72
references.pmid: 18843052
references.citation: Biochem J. 2014 Jul 15;461(2):315-22
references.pmid: 24785657
references.citation: Cell. 2013 Jan 17;152(1-2):290-303
references.pmid: 23332761
references.citation: EMBO Rep. 2009 Mar;10(3):285-92
references.pmid: 19180116
references.citation: J Cell Biol. 2010 Sep 6;190(5):881-92
references.pmid: 20819940
references.citation: Curr Protoc Cell Biol. 2012 Mar;Chapter 15:Unit 15.16
references.pmid: 22422474
references.citation: Biochim Biophys Acta. 2000 Jan 31;1490(1-2):189-97
references.pmid: 10786636
references.citation: J Biol Chem. 2005 Sep 23;280(38):33076-82
references.pmid: 16049009
references.citation: Mol Biol Cell. 2006 Apr;17(4):1527-39
references.pmid: 16421251
references.citation: Mol Cell Biol. 2009 Jan;29(1):157-71
references.pmid: 18936157
references.citation: Nature. 1999 Dec 9;402(6762):672-6
references.pmid: 10604474
references.citation: Nat Cell Biol. 2007 Oct;9(10):1142-51
references.pmid: 17891140
references.citation: J Biol Chem. 1996 Sep 27;271(39):24048-54
references.pmid: 8798641
references.citation: Nat Cell Biol. 2008 Jul;10(7):776-87
references.pmid: 18552835
references.citation: Nat Cell Biol. 2009 Apr;11(4):385-96
references.pmid: 19270696
references.citation: J Cell Biol. 2010 Nov 15;191(4):827-43
references.pmid: 21059846
references.citation: Autophagy. 2013 Oct;9(10):1491-9
references.pmid: 23884233
references.citation: J Cell Biol. 2013 Oct 28;203(2):299-313
references.pmid: 24165940
references.citation: Cell. 2007 Mar 9;128(5):931-46
references.pmid: 17350577
references.citation: Biochem J. 2008 Feb 15;410(1):1-17
references.pmid: 18215151
references.citation: Proc Natl Acad Sci U S A. 2012 Feb 7;109(6):2003-8
references.pmid: 22308354