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

• Main Authors: Malte Karow, Sarah Fischer, Susanne Meßling, Roman Konertz, Jana Riehl, Qiuhong Xiong, Ramesh Rijal, Prerana Wagle, Christoph S. Clemen, Ludwig Eichinger
• Format: Article
• Language: English
• Published: MDPI AG 2020-05-01
• Series: Cells
• Subjects: ATG12~5/16 complex; autophagy; Dictyostelium; ubiquitin-like protein; phagocytosis; pinocytosis
• Online Access: https://www.mdpi.com/2073-4409/9/5/1179

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. <i>Dictyostelium discoideum</i> <i>atg5</i> single, <i>atg5</i><i>/</i><i>12</i> double, and <i>atg5</i><i>/</i><i>12</i><i>/</i><i>16</i> 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 <i>Klebsiella aerogenes</i> was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but<i> </i>strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of <i>Escherichia coli</i> was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of <i>E. coli</i><i>,</i> 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.