Abstract
Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase β3 (CCTβ3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTβ3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTβ3 and is enhanced by its overexpression. This CCTβ3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTβ3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTβ3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival.
Highlights
Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive
These results indicate that activation of CTP:phosphocholine cytidylyltransferase β3 (CCTβ3) on autophagy-derived lipid droplets (LDs) is crucial for de novo synthesis of PC, which is used for autophagic membrane formation and thereby sustains autophagy for a long time
De novo-synthesized choline-containing phospholipids were labeled by culturing mouse embryo fibroblasts (MEFs) with propargylcholine (0.25 mM) for 1 h, followed by fixation and conjugation of fluorescent azide using the click reaction[19] (Supplementary Fig. 1A)
Summary
Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. As starvation is prolonged (e.g., 8 h), CCTβ3 is recruited to lipid droplets (LDs) generated from autophagic digests[17,18], and autophagic membranes emerge from the vicinity of those LDs. The CCTβ3 expression level is correlated with the autophagic activity only in cells starved for an extended period of time, and depletion of CCTβ3 in an osteosarcoma cell line, U2OS, reduces cell survival significantly. The CCTβ3 expression level is correlated with the autophagic activity only in cells starved for an extended period of time, and depletion of CCTβ3 in an osteosarcoma cell line, U2OS, reduces cell survival significantly These results indicate that activation of CCTβ3 on autophagy-derived LDs is crucial for de novo synthesis of PC, which is used for autophagic membrane formation and thereby sustains autophagy for a long time. CCTβ3 may be targeted to suppress prolonged autophagy in cancer cells in vivo
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