Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

• Published in: Cells (Basel, Switzerland) Switzerland), 2020-05, Vol.9 (5), p.1179
• Main Authors: Karow, Malte, Fischer, Sarah, Meßling, Susanne, Konertz, Roman, Riehl, Jana, Xiong, Qiuhong, Rijal, Ramesh, Wagle, Prerana, Clemen, Christoph S, Eichinger, Ludwig
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; ATG12~5/16 complex; Autophagy; Autophagy-Related Proteins - chemistry; Autophagy-Related Proteins - metabolism; Cell Proliferation; Cell Survival; Conserved Sequence; Dictyostelium; Dictyostelium - cytology; Dictyostelium - metabolism; Mutation - genetics; Phagocytosis; Phenotype; Pinocytosis; Proteostasis; Protozoan Proteins - chemistry; Protozoan Proteins - metabolism; Reproducibility of Results; ubiquitin-like protein
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells9051179

Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric ATG12~5/16 complex, which is part of the essential ubiquitin-like conjugation systems in autophagy. single, double, and triple gene knock-out mutant strains displayed similar defects in the conjugation of ATG8 to phosphatidylethanolamine, development, and cell viability upon nitrogen starvation. This implies that ATG5, 12 and 16 act as a functional unit in canonical autophagy. Macropinocytosis of TRITC dextran and phagocytosis of yeast were significantly decreased in ATG5¯ and ATG5¯/12¯ and even further in ATG5¯/12¯/16¯ cells. In contrast, plaque growth on was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of was seen for ATG5¯ and ATG5¯/12¯/16¯ cells. Proteasomal activity was also disturbed in a complex fashion, consistent with an inhibitory activity of ATG16 in the absence of ATG5 and/or ATG12. Our results confirm the essential function of the ATG12~5/16 complex in canonical autophagy, and furthermore are consistent with autophagy-independent functions of the complex and its individual components. They also strongly support the placement of autophagy upstream of the ubiquitin-proteasome system (UPS), as a fully functional UPS depends on autophagy.