Consideration about negative controls for LC3 and expression vectors for four colored fluorescent protein-LC3 negative controls

Autophagy. 2008 Jan;4(1):131-4. doi: 10.4161/auto.5233. Epub 2007 Nov 1.


A cytosolic form of LC3 is conjugated to phosphatidylethanolamine by Atg7, an E1-like enzyme, and Atg3, an E2-like enzyme, during autophagy. To monitor intracellular autophagosomes and autolysosomes, GFP-LC3 is a useful tool. However, GFP-LC3 can aggregate without being conjugated to phosphatidylethanolamine, especially when GFP-LC3 is transiently expressed (Kuma A, Matsui M, Mizushima N. Autophagy 2007; 3:323-8). Therefore, as a negative control, we investigated a mutant human LC3DeltaG protein in which the C-terminal Gly(120) essential for LC3-lipidation is deleted, and generated a set of expression plasmids for wild-type human LC3 and mutant LC3DeltaG fused to either CFP, GFP, YFP, or HcRed at the N terminus. We found that the mutant LC3DeltaG protein does not react with human Atg7 and Atg3, indicating that LC3-lipidation does not occur, and few puncta containing mutant LC3DeltaG form under starvation conditions. As observed with wildtype HcRed-LC3, mutant HcRed-LC3DeltaG also co-localizes with polyQ150-aggregates suggesting that the colocalization of HcRed- LC3 to polyQ150-aggregates is independent of LC3-lipidation. These mutant LC3DeltaG proteins will be useful negative controls in recognizing non-specific fluorescent protein-LC3 aggregates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy
  • Genetic Vectors* / genetics
  • Genetic Vectors* / metabolism
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Reference Values


  • Luminescent Proteins
  • MAP1LC3A protein, human
  • Microtubule-Associated Proteins
  • Recombinant Fusion Proteins