Mechanism of cargo-directed Atg8 conjugation during selective autophagy

Elife. 2016 Nov 23;5:e18544. doi: 10.7554/eLife.18544.

Abstract

Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.

Keywords: S. cerevisiae; biochemical reconstitution; biochemistry; cargo receptor; human; membrane biology; protein conjugation; selective autophagy.

MeSH terms

  • Amino Acid Sequence
  • Autophagy / genetics*
  • Autophagy-Related Protein 12 / genetics
  • Autophagy-Related Protein 12 / metabolism
  • Autophagy-Related Protein 5 / chemistry*
  • Autophagy-Related Protein 5 / genetics
  • Autophagy-Related Protein 5 / metabolism
  • Autophagy-Related Protein 8 Family / chemistry*
  • Autophagy-Related Protein 8 Family / genetics
  • Autophagy-Related Protein 8 Family / metabolism
  • Autophagy-Related Proteins / chemistry*
  • Autophagy-Related Proteins / genetics
  • Autophagy-Related Proteins / metabolism
  • Binding Sites
  • Biological Transport
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression Regulation
  • HeLa Cells
  • Humans
  • Molecular Docking Simulation
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Structure, Secondary
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequestosome-1 Protein / genetics
  • Sequestosome-1 Protein / metabolism
  • Signal Transduction
  • Transcription Factor TFIIIA / genetics
  • Transcription Factor TFIIIA / metabolism
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism

Substances

  • ATG12 protein, S cerevisiae
  • ATG16 protein, S cerevisiae
  • ATG19 protein, S cerevisiae
  • ATG5 protein, S cerevisiae
  • ATG8 protein, S cerevisiae
  • Atg34 protein, S cerevisiae
  • Autophagy-Related Protein 12
  • Autophagy-Related Protein 5
  • Autophagy-Related Protein 8 Family
  • Autophagy-Related Proteins
  • CALCOCO2 protein, human
  • Nuclear Proteins
  • OPTN protein, human
  • Receptors, Cell Surface
  • Receptors, Cytoplasmic and Nuclear
  • Recombinant Proteins
  • SQSTM1 protein, human
  • Saccharomyces cerevisiae Proteins
  • Sequestosome-1 Protein
  • Transcription Factor TFIIIA
  • Vesicular Transport Proteins