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The Evolutionarily Conserved Interaction Between LC3 and p62 Selectively Mediates Autophagy-Dependent Degradation of Mutant Huntingtin

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Abstract

Mammalian p62/sequestosome-1 protein binds to both LC3, the mammalian homologue of yeast Atg8, and polyubiquitinated cargo proteins destined to undergo autophagy-mediated degradation. We previously identified a cargo receptor-binding domain in Atg8 that is essential for its interaction with the cargo receptor Atg19 in selective autophagic processes in yeast. We, thus, sought to determine whether this interaction is evolutionally conserved from yeast to mammals. Using an amino acid replacement approach, we demonstrate that cells expressing mutant LC3 (LC3-K30D, LC3-K51A, or LC3-L53A) all exhibit defective lipidation of LC3, a disrupted LC3–p62 interaction, and impaired autophagic degradation of p62, suggesting that the p62-binding site of LC3 is localized within an evolutionarily conserved domain. Importantly, whereas cells expressing these LC3 mutants exhibited similar overall autophagic activity comparable to that of cells expressing wild-type LC3, autophagy-mediated clearance of the aggregation-prone mutant Huntingtin was defective in the mutant-expressing cells. Together, these results suggest that p62 directly binds to the evolutionarily conserved cargo receptor-binding domain of Atg8/LC3 and selectively mediates the clearance of mutant Huntingtin.

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Abbreviations

Atgs:

Autophagy-related proteins

DMP:

Dimethyl pimelimidate dihydrochloride

EBSS:

Earle’s buffered salt saline

GFP:

Green fluorescent protein

HD:

Huntington’s disease

HEK293:

Human embryonic kidney cells

Htt:

Huntingtin

LC3:

Microtubule-associated protein (MAP) light chain 3

PE:

Phosphatidylethanolamine

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Acknowledgments

We thank Drs. Nin-Nin Chuang, Jen-Leih Wu, and John Yu for generous support. The authors are also grateful to Dr. Yijuang Chern at Academia Sinica for providing the pcDNA3.1-Htt-(Q)109-hrGFP construct. The authors thank the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica for technical support. DNA sequencing analyses were carried out by the Core Facilities for Basic Genomic Research located at the National Yang-Ming University Genome Center. This study was supported by the National Science Council, Taiwan (grant number NSC 96-2311-B-002-012-MY3 to W.-P. H.) and Academia Sinica (to Y.-F.L.).

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Authors and Affiliations

  1. Institute of Cellular and Organismic Biology, Academia Sinica, ICOB 238, 128 Sec. 2 Academia Rd., Taipei, 11529, Taiwan

    Ying-Tsen Tung & Yung-Feng Liao

  2. Institute of Zoology, National Taiwan University, Taipei, Taiwan

    Ying-Tsen Tung, Hsinyu Lee & Wei-Pang Huang

  3. Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan

    Wen-Ming Hsu

  4. Department of Life Science, National Taiwan University, 1 Sec. 4 Roosevelt Rd., Taipei, Taiwan

    Hsinyu Lee & Wei-Pang Huang

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  1. Ying-Tsen Tung
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  2. Wen-Ming Hsu
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  3. Hsinyu Lee
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  4. Wei-Pang Huang
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  5. Yung-Feng Liao
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Correspondence to Wei-Pang Huang or Yung-Feng Liao.

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Tung, YT., Hsu, WM., Lee, H. et al. The Evolutionarily Conserved Interaction Between LC3 and p62 Selectively Mediates Autophagy-Dependent Degradation of Mutant Huntingtin. Cell Mol Neurobiol 30, 795–806 (2010). https://doi.org/10.1007/s10571-010-9507-y

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