Novel Insights into the Cellular Localization and Regulation of the Autophagosomal Proteins LC3A, LC3B and LC3C

Abstract

Macroautophagy is a conserved degradative process for maintaining cellular homeostasis and plays a key role in aging and various human disorders. The microtubule-associated protein 1A/1B light chain 3B (MAP1LC3B or LC3B) is commonly analyzed as a key marker for autophagosomes and as a proxy for autophagic flux. Three paralogues of the LC3 gene exist in humans: LC3A, LC3B and LC3C. The molecular function, regulation and cellular localization of LC3A and LC3C have not been investigated frequently, even if a similar function to that described for LC3B appears likely. Here, we have selectively decapacitated LC3B by three separate strategies in primary human fibroblasts and analyzed the evoked effects on LC3A, LC3B and LC3C in terms of their cellular distribution and co-localization with p62, a ubiquitin and autophagy receptor. First, treatment with pharmacological sirtuin 1 (SIRT1) inhibitors to prevent the translocation of LC3B from the nucleus into the cytosol induced an increase in cytosolic LC3C, a heightened co-localization of LC3C with p62, and an increase LC3C-dependent autophagic flux as assessed by protein lipidation. Cytosolic LC3A, however, was moderately reduced, but also more co-localized with p62. Second, siRNA-based knock-down of SIRT1 broadly reproduced these findings and increased the co-localization of LC3A and particularly LC3C with p62 in presumed autophagosomes. These effects resembled the effects of pharmacological sirtuin inhibition under normal and starvation conditions. Third, siRNA-based knock-down of total LC3B in cytosol and nucleus also induced a redistribution of LC3C as if to replace LC3B in the nucleus, but only moderately affected LC3A. Total protein expression of LC3A, LC3B, LC3C, GABARAP and GABARAP-L1 following LC3B decapacitation was unaltered. Our data indicate that nuclear trapping and other causes of LC3B functional loss in the cytosol are buffered by LC3A and actively compensated by LC3C, but not by GABARAPs. The biological relevance of the potential functional compensation of LC3B decapacitation by LC3C and LC3A warrants further study.

The selective degradation of sirtuins via macroautophagy in the MPP+ model of Parkinson’s disease is promoted by conserved oxidation sites

“…To verify whether SIRTs actually co-localize with autophagosomal structures, we performed immunocytochemistry for SIRT1, SIRT3, SIRT4, SIRT5, and LC3B. Analysis was restricted to cytosolic co-localization to exclude nuclear co-localization not related to autophagosomes [ 14 ]. Co-localization between cytosolic SIRTs and LC3B was significantly induced in MPP + - and BafA1-treated cells (versus MPP + -alone treated cells), suggesting higher incorporation of SIRTs into autophagosomal structures (Fig.…”

“…Immunoblotting was performed according to published protocols [ 14 , 18 ]. Primary antibodies (1:1000) were detected by horseradish peroxidase-conjugated secondary antibodies (Dianova, 1:10000) through luminescence and quantified using densitometry.…”

“…Immunocytochemistry and co-localization analysis were performed according to published protocols [ 14 , 18 ]. Primary antibodies (1:200) were labeled with secondary antibodies (Dianova, 1:400).…”

“…SIRTs indeed do contribute to autophagy by directly regulating different components of the autophagic machinery through deacylation. For example, SIRT1 reportedly regulates cytosolic availability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B, also known as LC3 or LC3B), an autophagosomal component of major importance [ 14 , 15 ]. Similar observations were published in vivo concerning the acetylation status of ATG5 and ATG7 [ 16 ].…”

Autophagy Markers p62, LC3II and Beclin1 Correlate with Clark Levels in Melanoma Tumors

“…LC3 (microtubuleassociated protein 1A/1B-light chain 3) comprises of a solubleLC3 I and a lipidated form, called LC3 II. LC3 II is involved in autophagy and recruited into autophagosomes [11]. Different types of stressors promote the conjugation of LC3 I tophosphatidylethanolamine, to organize the autophagosome speci c LC3 II, which is considered the most reliablemarker of autophagy [12].…”