Regulation of macroautophagy and microautophagic lipophagy by phosphatidylserine synthase Cho1 and external ethanolamine

Abstract Phospholipids play crucial roles in autophagy; however, the underlying mechanisms remain elusive. We previously found that the phosphatidylserine (PtdSer) transporter Osh5 is critical for autophagosome formation. Therefore, in this study, we aimed to investigate the impact of the knockout of cho1, which encodes PtdSer synthase, on autophagy. Green fluorescent protein-autophagy-related gene 8 (GFP-Atg8) processing assay revealed a significant defect in the macroautophagic activity of the cho1Δ mutant, regardless of the presence or absence of ethanolamine (Etn). Notably, autophagosomes were absent in the cytosol, and macroautophagic bodies were not observed in the vacuoles of the starved cho1Δ mutant, underscoring the essential role of PtdSer synthesized using Cho1 in autophagosome biogenesis. In contrast, numerous microautophagic vesicles containing lipid droplets were observed in the vacuoles of cho1Δ mutants starved in the presence of Etn, suggesting the crucial role of phosphatidylethanolamine (PtdEtn) synthesized via the Kennedy pathway in microautophagic lipophagy when PtdSer synthesis using Cho1 is disrupted. Given recent evidence pointing to the involvement of the ubiquitination system in various autophagy-related processes, we also examined the role of ubiquitin-conjugating enzyme E2 gene ubc4. Moreover, microautophagic lipophagy was significantly diminished in starved cho1Δ yeast with ubc4 knockout. These findings suggest the critical role of Ubc4-mediated ubiquitination in facilitating microautophagic lipophagy of the vacuole in yeast cells. Competing Interest Statement The authors have declared no competing interest. Footnotes The author order has been revised. Abbreviations - BSA - bovine serum albumin - CDP - cytidine diphosphate - DAG - diacylglycerol - EF - E-face (exoplasmic leaflet) - EM - electron microscopy - ESCRT - endosomal sorting complex required for transport - Etn - ethanolamine - GFP - green fluorescent protein - GST - glutathione S-transferase - HRP - horse radish peroxidase - IMP - intra-membrane particle - ORF - open reading frame - Osh - oxysterol-binding protein homologue - PBS - phosphate-buffered saline - PF - P-face (protoplasmic or cytoplasmic leaflet) - PH - pleckstrin homology - PtdEtn - phosphatidylethanolamine - PtdSer - phosphatidylserine - PtdIns(3)P - phosphatidylinositol 3-phosphate - PtdIns(4)P - phosphatidylinositol 4-phosphate - PtdIns(4,5)P2 - phosphatidylinositol 4,5-bisphosphate - QF - quick-freezing - QF-FRL - quick-freezing and freeze-fracture replica labeling - RT-PCR - reverse transcription-polymerase chain reaction - SDS - sodium dodecyl sulfate - YPD - yeast peptone dextrose - WT - wild-type