The role of phospholipids in autophagosome formation
Eukaryotic cells have the unique ability to remove or recycle organelles within the cell through double membrane structures called autophagosomes. To date, a large debate has emerged in explaining where the membrane of the autophagosomes arises. The CDP-ethanolamine (Kennedy) pathway produces phosphatidylethanolamine (PE), an essential phospholipid in the inner membrane of the cell. This thesis demonstrates in liver cells that the CDP-ethanolamine pathway is upregulated during amino acid deprivation, a strong activator of autophagy. Through immunoprecipitation and radiolabelling with [14C] ethanolamine, it was determined that PE synthesized from the CDP-ethanolamine pathway lipidates LC3-I, to form LC3-II, a biomarker for autophagy. Through pulse-chase experiments with [14C] ethanolamine and [3H] glycerol, it was shown that PE was more utilized throughout the starvation state, unlike other phospholipids phosphatidylserine (PS) and phosphatidylcholine (PC). In addition, during upregulated autophagy, there is an increase in synthesis of all phospholipids (PE, PC and PS). Interestingly, there was no change in diacylglycerol levels between the starvation and nutrient rich state. Meanwhile, triacylglycerol levels showed an increased rate of degradation during the starvation state, suggesting that the donation of the fatty acids for phospholipid synthesis was due to triacylglycerol metabolism, and not from diacylglycerol. It was concluded that PE made by the CDP-ethanolamine pathway may be a critical component for autophagosome formation.