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The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans

Abstract

In Caenorhabditis elegans embryos, paternally provided organelles, including mitochondria, are eliminated by a process of selective autophagy called allophagy, the mechanism by which mitochondrial DNA is inherited maternally. However, it remains unclear how paternal organelles are recognized and targeted for autophagy. Here, we identified an autophagy receptor for allophagy, ALLO-1. ALLO-1 is essential for autophagosome formation around paternal organelles and directly binds to the worm LC3 homologue LGG-1 through its LC3-interacting region (LIR) motif. After fertilization, ALLO-1 accumulates on the paternal organelles before autophagosome formation, and this localization depends on the ubiquitin modification of the paternal organelles. We also identified IKKE-1, a worm homologue of the TBK1 and IKKε family kinase, as another critical regulator of allophagy. IKKE-1 interacts with ALLO-1, and the IKKE-1-dependent phosphorylation of ALLO-1 is important for paternal organelle clearance. Thus, we propose that ALLO-1 is the allophagy receptor whose function is regulated by IKKE-1-dependent phosphorylation.

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Fig. 1: Identification of IKKE-1 and ALLO-1 as allophagy regulators.
Fig. 2: Allophagy is impaired in ikke-1 and allo-1 mutants.
Fig. 3: IKKE-1 and ALLO-1 are maternally expressed and localize to paternal organelles.
Fig. 4: ALLO-1 and IKKE-1 localizations precede LGG-1 recruitment.
Fig. 5: ALLO-1 physically interacts with IKKE-1.
Fig. 6: ALLO-1 physically interacts with LGG-1 and regulates LGG-1 recruitment.
Fig. 7: IKKE-1-dependent phosphorylation of ALLO-1 is important for allophagy.
Fig. 8: ALLO-1-mediated autophagosome formation depends on ubiquitylation.

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Acknowledgements

We thank S. Tomizawa and the members of the Sato laboratory for technical assistance and discussions, N. Mizushima (The University of Tokyo, Japan) for discussions, S. Mitani (Tokyo Women’s Medical University, Japan) and the Caenorhabditis Genetic Center for supplying the C. elegans strains, N. Matsuda (Tokyo Metropolitan Institute of Medical Science, Japan) and K. Honma (Maebashi Institute of Technology, Japan) for technical advice and Y. Kohara (National Institute of Genetics, Japan), A. Audhya (University of Wisconsin-Madison, USA) and B. Grant (Rutgers University, USA) for the plasmid and antibody. This research was supported by the MEXT KAKENHI (grant numbers 26111503 and 16H01191), The Cell Science Research Foundation, the Uehara Memorial Foundation, Takeda Science Foundation and Joint Usage and Joint Research Programs of the Institute of Advanced Medical Sciences, Tokushima University (to M.S.), and by the JSPS KAKENHI (grant numbers 26291036, 17K19377 and 17H03669), Sumitomo Foundation, Naito Foundation and Ono Medical Research Foundation (to Ken S.). This work was also supported by the Joint Research Program of the Institute for Molecular and Cellular Regulation at Gunma University.

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M.S. and Ken S. designed the experiments and analysed the data. M.S., Katsuya S. and K.T. performed the experiments. H.K. performed LC-MS/MS analysis. M.S., Ken S. and H.K. wrote the manuscript.

Corresponding authors

Correspondence to Miyuki Sato or Ken Sato.

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Supplementary information

Supplementary Information

Supplementary Figures 1–6, Supplementary Table Legends.

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Supplementary Table 1

Identification of proteins copurified with GFP-ALLO-1 by LC-MS/MS analysis. List of top 20 peptides copurified with GFP or GFP-ALLO-1. Number of peptides found in LC-MS/MS analysis is shown. Experiment was repeated three times with similar results.

Supplementary Table 2

List of kinases screened in this study. The genes predicted to encode kinases were selected from the genome-wide RNAi library (360 genes).

Supplementary Table 3

List of antibodies used in this study.

Supplementary Table 4

C. elegans strain list used in this study.

Supplementary Table 5

Statistics source data supporting Figures 6b, 7f and 8g.

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Sato, M., Sato, K., Tomura, K. et al. The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans . Nat Cell Biol 20, 81–91 (2018). https://doi.org/10.1038/s41556-017-0008-9

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