LC3C contributes to autophagosome elongation/fusion in autophagy phenomena

Autophagy is a conserved cellular process through which superfluous or damaged material is recycled. One of the first steps in autophagy is the formation of a double- membrane structure, called autophagosome, which engulfs the material to be degraded. The generation of this structure is a complex event, in which many proteins are involved. Among the latter, yeast Atg8 or its mammalian orthologues are essential in autophagosome membrane elongation, shaping and closure. A subfamily of the human Atg8 orthologues is formed by the proteins LC3A, LC3B, and LC3C. Previous studies suggest that, at variance with the other two, LC3C does not participate in cardiolipin- mediated mitophagy. The present study was devoted to exploring the binding of LC3C to lipid vesicles, bilayers and monolayers, and the ensuing protein-dependent perturbing effects, in the absence of the mitochondrial lipid cardiolipin. All Atg8 orthologues are covalently bound to a phospholipid prior to their involvement in autophagosome elongation. In our case, a mutant in the C-terminal amino acid, LC3C G126C, together with the use of a maleimide-derivatized phosphatidyl ethanolamine, ensured LC3C lipidation. Ultracentrifugation, surface pressure measurements, spectroscopic and cryo- electron microscopic techniques revealed that lipidated LC3C induced vesicle aggregation and inter-vesicular lipid mixing, including inner-monolayer lipid mixing, consistent with in vitro vesicle-vesicle fusion. LC3C was also able to cause the release of vesicular aqueous contents. The data support the idea that LC3C would be able to help in autophagosome elongation/fusion in autophagy phenomena.