Phosphorylation of Atg12 for optimal autophagy in yeast <i>Komagataella phaffii</i>

Macroautophagy is an evolutionarily conserved degradation pathway in eukaryotes that mediates the turnover of cytoplasmic components. The formation of autophagosomes, a hallmark of autophagy, involves autophagy-related (Atg) proteins, including two ubiquitin-like conjugation systems, Atg12 system and Atg8 system. In most species, Atg12 covalently binds Atg5, forming the Atg12-Atg5-Atg16 complex that functions as an E3-like enzyme to promote Atg8 conjugation with phosphatidylethanolamine (PE), a step essential for autophagosomal maturation. By contrast, certain species such as yeast Komagataella phaffii lack Atg10 and/or the C-terminal glycine of Atg12, relying instead on a non-covalent Atg12-Atg5 complex. However, the physiological significance of this reductively evolved non-covalent system and its divergence in molecular mechanisms from species harbouring the canonical covalent Atg12 system remain undiscussed. In this study, we demonstrate that under nitrogen starvation, KpAtg12 is phosphorylated and lipidation of KpAtg8 is enhanced. Our results with a phosphorylation-deficient mutant of KpAtg12 suggest that KpAtg12 phosphorylation modulates the activity of nitrogen starvation-induced macroautophagy through KpAtg8 lipidation reaction.