Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs

Exp Cell Res. 2015 Apr 10;333(1):21-38. doi: 10.1016/j.yexcr.2015.02.003. Epub 2015 Feb 13.

Abstract

LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.

Keywords: Autophagy; Density gradient; GABARAP; LC3 antibody; Sequestration; Subcellular fractionation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy*
  • Autophagy-Related Protein 5
  • Cells, Cultured
  • Male
  • Mice
  • Microtubule-Associated Proteins / metabolism*
  • Microtubule-Associated Proteins / physiology*
  • Proteins / metabolism
  • Rats, Wistar
  • Vacuoles / metabolism

Substances

  • Atg5 protein, rat
  • Autophagy-Related Protein 5
  • GABARAP protein, rat
  • LC3 protein, rat
  • Microtubule-Associated Proteins
  • Proteins