Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autophagosomal membranes

J Biol Chem. 2004 Nov 12;279(46):47704-10. doi: 10.1074/jbc.M407016200. Epub 2004 Sep 7.

Abstract

Human light chain 3/MAP1LC3B, an autophagosomal ortholog of yeast Atg8, is conjugated to phospholipid (PL) via ubiquitylation-like reactions mediated by human Atg7 and Atg3. Since human Atg4B was found to cleave the carboxyl terminus of MAP1LC3B in vitro, we hypothesized that this exposes its carboxyl-terminal Gly(120). It was recently reported, however, that when Myc-MAP1LC3B-His is expressed in HEK293 cells, its carboxyl terminus is not cleaved. (Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., and Kominami, E. (2004) J. Biol. Chem. 279, 36268-36276). To clarify this contradiction, we sought to determine whether the carboxyl terminus of MAP1LC3B is cleaved to expose Gly(120) for further ubiquitylation-like reactions. When MAP1LC3B-3xFLAG and Myc-MAP1LC3B-His were expressed in HEK293 cells, their carboxyl termini were cleaved, whereas there was little cleavage of mutant proteins MAP1LC3B(G120A)-3xFLAG and Myc-MAP1LC3B(G120A)-His, containing Ala in place of Gly(120). An in vitro assay showed that Gly(120) is essential for carboxyl-terminal cleavage by human Atg4B as well as for formation of the intermediates Atg7-MAP1LC3B (ubiquitin-activating enzyme-substrate) and Atg3-MAP1LC3B (ubiquitin carrier protein-substrate). Recombinant MAP1LC3B-PL was fractionated into the 100,000 x g pellet in a manner similar to that shown for endogenous MAP1LC3B-PL. RNA interference of MAP1LC3B mRNA resulted in a decrease in both endogenous MAP1LC3B-PL and MAP1LC3B. These results indicate that the carboxyl terminus of MAP1LC3B is cleaved to expose Gly(120) for further ubiquitylation-like reactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Autophagy / physiology*
  • Autophagy-Related Protein 7
  • Cell Fractionation
  • Cell Line
  • Glycine / metabolism*
  • Humans
  • Intracellular Membranes / metabolism*
  • Methionine / metabolism*
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Molecular Sequence Data
  • Phospholipids / metabolism*
  • Protein Structure, Tertiary
  • RNA Interference
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Alignment
  • Subcellular Fractions / metabolism

Substances

  • ATG7 protein, S cerevisiae
  • MAP1LC3A protein, human
  • MAP1LC3B protein, human
  • MAP1LC3C protein, human
  • Microtubule-Associated Proteins
  • Phospholipids
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Methionine
  • Autophagy-Related Protein 7
  • Glycine