Functional Characterization of Ubiquitin-Like Core Autophagy Protein ATG12 in Dictyostelium discoideum

Autophagyis a highly conserved intracellular degradative pathway that is crucial for cellular homeostasis. During autophagy, the core autophagyprotein ATG12plays, together with ATG5and ATG16, an essential role in the expansion of the autophagosomalmembrane. In this study we analyzed gene replacement mutants of atg12in Dictyostelium discoideumAX2 wild-type and ATG16‾ cells. RNAseq analysis revealed a strong enrichment of, firstly, autophagygenes among the up- regulated genesand, secondly, genes implicated in cell motility and phagocytosisamong the down- regulated genesin the generated ATG12‾, ATG16‾ and ATG12‾/ 16‾ cells. The mutant strains showed similar defects in fruiting body formation, autolysosome maturation, and cellular viability, implying that ATG12and ATG16 act as a functional unit in canonical autophagy. In contrast, ablation of ATG16 or of ATG12and ATG16 resulted in slightly more severe defects in axenicgrowth, macropinocytosis, and protein homeostasis than ablation of only ATG12, suggesting that ATG16 fulfils an additional functionin these processes. Phagocytosisof yeast, spore viability, and maximal cell density were much more affected in ATG12‾/ 16‾ cells, indicating that both proteins also have cellular functions independent of each other. In summary, we show that ATG12and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy.