The association of the ATG12~5 conjugate with ATG16 unmasks a membrane-binding site in ATG5 and the membrane tethering ability of ATG5 is also stimulated by ATG12 . unit in canonical autophagy. In contrast, ablation of ATG16 or of ATG12 and ATG16 resulted in slightly more severe defects in axenic growth, macropinocytosis, and protein homeostasis than ablation of only ATG12, suggesting that ATG16 fulfils an additional function in these processes. Phagocytosis of yeast, spore viability, and maximal cell density were much more affected in ATG12/16 cells, indicating that both proteins also have cellular functions impartial of each other. In summary, we show that ATG12 and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy. . The proteins involved in autophagosome formation were named ATG, for AuTophaGy-related proteins, and are evolutionarily highly conserved across the eukaryotic lineage [7,8]. Autophagic dysfunction can result in a wide range of diseases, including neurodegeneration, malignancy, muscular dystrophy, and lipid-storage disorders [3,9]. The autophagic process can be subdivided into initiation, maturation, and lysosomal degradation phases. In the initiation phase, the so-called omegasome (phagophore assembly site or PAS in . Its 3D structure is similar to the structure of ubiquitin and is highly conserved from yeast to man. ATG12 proteins from different organisms share a so-called APG12 domain name which Gpc2 shows the conserved ubiquitin-fold in the crystal structure  (Physique 1B). The APG12 domain name is required for both MC-VC-PABC-Aur0101 the conjugation to ATG5 and canonical autophagy . ATG12 is usually part of the heterotetrameric ATG12~5/16 complex which localizes to the outer membrane of the expanding isolation membrane and is released shortly before or after autophagosome completion . The association of the ATG12~5 conjugate with ATG16 unmasks a membrane-binding site in ATG5 and the membrane tethering ability of ATG5 is also stimulated by ATG12 . Within the ATG12~5/16 complex, ATG16 is required for correct localization and the ATG12~5 conjugate possesses E3 ligase activity that promotes the conjugation of ATG8 to PE at the autophagic membrane [17,21,22]. Knock-out mutants of ATG12 have shown postnatal lethality in mice and are not able to form cysts and fruiting body in Ascomyceta and Amoebozoa [23,24,25,26]. However, despite extensive study, the precise cellular functions of ATG12 are still not fully comprehended. The interpersonal amoeba is usually a well-established model organism used to study the autophagic process . Under nutrient-rich conditions, cells grow as unicellular amoebae that divide by binary cell fission and feed on bacteria by phagocytosis . Upon depletion of the food source, solitary amoebae aggregate and undergo distinct morphological says, giving MC-VC-PABC-Aur0101 rise to mature fruiting body . Since the developmental stage takes place in the absence of nutrients, cells mobilize a large fraction of the required energy for morphogenesis and biosynthetic pathways by autophagy . Here we describe the consequences of the deletion of in AX2 wild-type and ATG16 cells for genome-wide transcription, development, autolysosome formation, growth, phagocytosis, macropinocytosis, and protein homeostasis. Our results reveal massive transcriptional changes and complex phenotypes of varying severity for the different knock-out strains, implying that ATG12 and ATG16 have, in addition to their role in canonical autophagy, autophagy-independent functions. Moreover, we could detect ATG12 only in the ATG12~5 conjugate and found no MC-VC-PABC-Aur0101 evidence for unconjugated ATG12. Our results also support links between autophagy and the uptake of nutrients as well as between autophagy and the ubiquitin-proteasome system (UPS). 2. Materials and Methods 2.1. Dictyostelium Strains, Growth, and Development AX2 was used as wild-type strain. The ATG12 and ATG12/16 strains were generated by replacement of the gene with the knock-out construct in AX2 and ATG16 cells . Strains expressing RFP-ATG12 or RFP-GFP-ATG8a were generated by transformation of AX2 and knock-out strains, respectively, with MC-VC-PABC-Aur0101 appropriate expression constructs as explained below. The strains used in this study are outlined in Table 1. All strains were produced at 22 C in liquid nutrient medium on plates (10 cm diameter) or with shaking at 160 rpm  or on as well as cell survival upon nitrogen starvation and development experiments were carried out as explained [32,37]. Development was analyzed at specific time points using a stereomicroscope (M205 C, Leica, Wetzlar, Germany) and the accompanying Leica LAS X software (v.3.3.0). To assess spore viability, spore balls.
The association of the ATG12~5 conjugate with ATG16 unmasks a membrane-binding site in ATG5 and the membrane tethering ability of ATG5 is also stimulated by ATG12