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

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 cho1∆ 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 cho1∆ 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 cho1∆ 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. In addition, deletion of ubc4 gene led to a pronounced reduction in microautophagic lipophagy in starved cho1∆ cells, but not in wild-type cells. Together, these observations highlight an essential role for Ubc4-mediated ubiquitination in driving vacuolar microautophagic lipophagy specifically under Cho1-deficient conditions.