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Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages

Abstract

Beclin 1, a protein essential for autophagy, binds to hVps34/Class III phosphatidylinositol-3-kinase and UVRAG. Here, we have identified two Beclin 1 associated proteins, Atg14L and Rubicon. Atg14L and UVRAG bind to Beclin 1 in a mutually exclusive manner, whereas Rubicon binds only to a subpopulation of UVRAG complexes; thus, three different Beclin 1 complexes exist. GFP–Atg14L localized to the isolation membrane and autophagosome, as well as to the ER and unknown puncta. Knockout of Atg14L in mouse ES cells caused a defect in autophagosome formation. GFP–Rubicon was localized at the endosome/lysosome. Knockdown of Rubicon caused enhancement of autophagy, especially at the maturation step, as well as enhancement of endocytic trafficking. These data suggest that the Beclin 1–hVps34 complex functions in two different steps of autophagy by altering the subunit composition.

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Figure 1: Identification and analysis of Beclin 1-binding proteins.
Figure 2: The effects of Atg14L and Rubicon knockdown on GFP–LC3 dot formation.
Figure 3: The effect of Atg14L knockout on protein degradation and LC3 turnover.
Figure 4: The effect of Atg14L knockout on autophagosome formation.
Figure 5: Localization of GFP–Atg14L.
Figure 6: The effects of Rubicon knockdown on autophagy.
Figure 7: Localization of Rubicon.
Figure 8: The role of Rubicon in autophagosome maturation and endocytic traffic.

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Acknowledgements

The authors thank Beth Levine for Beclin 1 cDNA, Noboru Mizushima for anti-Atg16L antibody and Atg5 knockout mouse ES cells, Sumio Sugano and Yutaka Suzuki for UVRAG cDNA, Toshio Kitamura for PLAT-E cells and pMX-puro vector, Roger Y. Tesien for the plasmid encoding mStrawberry protein, Ryuichi Masaki for pEGFP–msALDH(35) vector, Yusuke Yamada for Beclin 1 deletion mutants cDNA construction, Asaya Nishi and Kunihiro Kawanishi for technical assistance. The work described in this report was supported in part by Special Coordination Funds for Promoting Science and Technology of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

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K.M. performed most of the experiments; T. Saitoh, T. Satoh and S.A. generated Atg14−/− ES cells; K.T. performed the experiments shown in Fig. 8a, c and Supplementary Information , Fig. S5i; H.O. performed electron microscopy; N.K. performed the experiments shown in Supplementary Information, Fig. 3e and S5d; I.M. and K.S.N. provided technical support; T. Ichimura and T. Isobe provided MEF system techniques and mass spectrometry data analysis; K.M., T.N. and T.Y. analysed and discussed the data; T.N. and T. Y. wrote the manuscript; T. Y. supervised the project.

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Correspondence to Tamotsu Yoshimori.

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Matsunaga, K., Saitoh, T., Tabata, K. et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat Cell Biol 11, 385–396 (2009). https://doi.org/10.1038/ncb1846

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