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. 2010 Aug 23;190(4):511-21.
doi: 10.1083/jcb.200911141. Epub 2010 Aug 16.

Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L

Kohichi Matsunaga  1 , Eiji Morita, Tatsuya Saitoh, Shizuo Akira, Nicholas T Ktistakis, Tetsuro Izumi, Takeshi Noda, Tamotsu Yoshimori
Affiliations

Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L

Kohichi Matsunaga et al. J Cell Biol. .

Abstract

Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.

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Figures

Figure 1.
Figure 1.
Involvement of Atg14L in the initial phase of autophagosome formation. (a) HEK293 cells stably expressing GFP-DFCP1 were transfected with or without shRNA against Atg14L. The cell lysates were subjected to immunoblotting with the indicated antibodies. (b) GFP signals were observed in these cells before or after starvation. (c) The number of GFP-positive puncta per cell was counted; mean ± SD values are presented. N, nutrient rich; S, starvation. (d) HEK293 cells were lysed and subjected to subcellular fractionation as described in Materials and methods. Equivalent amounts of total, cytosolic, and ER fractions were subjected to immunoblotting with each organelle marker antibody. The percentage of recovery in each fraction is presented as mean ± SD values. (e) HEK293 cells stably expressing GFP-DFCP1 were transiently transformed with Flag-tagged Atg14L and subjected to starvation. The cells were fixed and stained with the indicated antibodies. Insets show higher magnification of the boxed areas. (f) HEK293 cells were transfected with GFP-Atg14L with or without mCherry-linked dominant-negative ULK1, and these cells were cultured in complete or starvation medium for 60 min. Bars, 10 µm.
Figure 2.
Figure 2.
Domain analysis of Atg14L structure. (a) HEK293 cells were simultaneously transfected with four plasmids harboring One-Strep-Flag (OSF)–tagged Atg14L, Beclin-1, hVps34, and hVps15, and Atg14L was pulled down using Strep-Tactin Sepharose beads. Empty plasmid was used as control instead of Atg14L. The precipitates were subjected to SDS-PAGE and Coomassie brilliant blue staining. (b) Diagram of Atg14L deletion mutant construction. (c) HEK293 cells transiently expressing each construct were subjected to coimmunoprecipitation analysis with the anti-GFP antibody. Each precipitant was subjected to immunoblotting with the indicated antibodies. IP, immunoprecipitation. (d) Alignment of N-terminal conserved region of Atg14 orthologues. Conserved cysteine repeats are boxed. The degree of sequence conservation is denoted: asterisks indicate a perfect match among interspecies, periods indicate mostly conserved, and colons indicate more conserved than the periods. Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), Danio rerio (Dr), Drosophila melanogaster (Dm), Candida glabrata (Cg), and Saccharomyces cerevisiae (Sc) are shown. (e) NRK cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14L4C4A and fixed under nutrient-rich conditions. The cells were stained with anticalnexin antibody as an ER membrane marker. Higher magnification of the boxed areas is shown below. Bars, 10 µm.
Figure 3.
Figure 3.
Ectopic expression of Atg14L in the plasma membrane. (a) A549 cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14LCAAX and subjected to starvation. The cells were fixed and stained with anti–Beclin-1 antibody. Higher magnification of the boxed areas is shown below. (b) HEK293 cells were cotransfected with MEF-hVps34 and GFP or GFP-Atg14LCAAX and stained with anti-Flag antibody. Bars, 10 µm.
Figure 4.
Figure 4.
Autophagy induction by overexpressed Atg14L. A549 cells were transiently transfected with adenovirus harboring GFP-Atg14L or GFP-Atg14L4C4A. (a and b) The cells before starvation (a) or after starvation (b) were fixed and stained with anti-LC3 antibody. (c) The number of LC3-positive puncta per cell was counted, and mean ± SD values are presented. Bars, 10 µm.
Figure 5.
Figure 5.
The effect of Atg14L localization on its function. (a) Atg14L knockout mouse ES cells were transfected with GFP, GFP-Atg14L, GFP-Atg14L4C4A, or GFP-ER-Atg14L4C4A of the pCAG vector and subjected to starvation. The cells were fixed and immunostained with anti-Atg16L or anti-LC3 antibodies. (b and c) The number of Atg16L-positive puncta (b) and LC3-positive puncta (c) per cell was counted in at least 10 GFP-positive cells, and mean ± SD values are presented. Bars, 10 µm.
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References

    1. Axe E.L., Walker S.A., Manifava M., Chandra P., Roderick H.L., Habermann A., Griffiths G., Ktistakis N.T. 2008. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182:685–701 10.1083/jcb.200803137 - DOI - PMC - PubMed
    1. Bozidis P., Williamson C.D., Colberg-Poley A.M. 2007. Isolation of endoplasmic reticulum, mitochondria, and mitochondria-associated membrane fractions from transfected cells and from human cytomegalovirus-infected primary fibroblasts. Curr. Protoc. Cell Biol. Chapter 3:Unit 3.27 10.1002/0471143030.cb0327s37 - DOI - PubMed
    1. Cheung P.C., Trinkle-Mulcahy L., Cohen P., Lucocq J.M. 2001. Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi. Biochem. J. 355:113–121 10.1042/0264-6021:3550113 - DOI - PMC - PubMed
    1. Fujita N., Hayashi-Nishino M., Fukumoto H., Omori H., Yamamoto A., Noda T., Yoshimori T. 2008a. An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol. Biol. Cell. 19:4651–4659 10.1091/mbc.E08-03-0312 - DOI - PMC - PubMed
    1. Fujita N., Itoh T., Omori H., Fukuda M., Noda T., Yoshimori T. 2008b. The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol. Biol. Cell. 19:2092–2100 10.1091/mbc.E07-12-1257 - DOI - PMC - PubMed

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