Mouse Apg10 as an Apg12-conjugating enzyme: analysis by the conjugation-mediated yeast two-hybrid method

FEBS Lett. 2002 Dec 18;532(3):450-4. doi: 10.1016/s0014-5793(02)03739-0.

Abstract

Autophagosome formation is a central event in macroautophagy. The Apg12-Apg5 conjugate, which is essential in this process, is generated by a ubiquitin-like protein conjugation system. In yeast, Apg12, following activation by the E1-like Apg7, forms a thioester with Apg10 (E2-like). Apg12 is finally conjugated to Apg5 via an isopeptide bond. The possible requirement of an E3-like protein for the conjugation, however, has not yet been confirmed. The Apg12 system is conserved among eukaryotes, although a mammalian counterpart of Apg10 has not yet been identified. Here, we report the identification and characterization of the mouse Apg10 ortholog. A yeast two-hybrid screen using the mouse Apg5 (mApg5) as bait identified a novel protein with 19% identity to yeast Apg10. We designated this protein mouse Apg10 (mApg10). We demonstrated by a modified yeast two-hybrid assay that mApg10 mediates the conjugation of mApg12 and mApg5. The in vivo interaction of mApg12 with mApg10 in HeLa cells suggests that mApg10 is an Apg12-conjugating enzyme, likely serving as an Apg5-recognition molecule in the Apg12 system. This novel two-hybrid method, which we have named 'conjugation-mediated yeast two-hybrid', proves to be a simple and useful technique with which to analyze protein-protein conjugation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Autophagy-Related Protein 12
  • Autophagy-Related Protein 5
  • Cloning, Molecular
  • DNA, Complementary / metabolism
  • Fungal Proteins / metabolism*
  • Fungal Proteins / physiology
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Mice
  • Molecular Sequence Data
  • Phagocytosis
  • Plasmids / metabolism
  • Protein Binding
  • Proteins / metabolism*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Transfection
  • Two-Hybrid System Techniques
  • Ubiquitin-Protein Ligases

Substances

  • ATG12 protein, S cerevisiae
  • ATG5 protein, S cerevisiae
  • Apg10 protein, S cerevisiae
  • Atg12 protein, mouse
  • Autophagy-Related Protein 12
  • Autophagy-Related Protein 5
  • DNA, Complementary
  • Fungal Proteins
  • Proteins
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin-Protein Ligases