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

Abstract

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. Dictyostelium discoideum atg5 single, atg5/12 double, and atg5/12/16 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 Klebsiella aerogenes 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 Escherichia coli was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of E. coli, 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.

The Intraflagellar Transport Protein IFT20 Recruits ATG16L1 to Early Endosomes to Promote Autophagosome Formation in T Cells

“…However, we have detected minimal co-localization of BECLIN 1 with the Golgi in T cells. As an alternative possibility, Golgi-associated ATG16L1 may play autophagy-independent functions in these cells, as recently reported in Dictyostelium discoideum ( Karow et al, 2020 ).…”

The intraflagellar transport protein IFT20 recruits ATG16L1 to early endosomes to promote autophagosome formation in T cells

“…However, we have detected minimal co-localization of BECLIN 1 with the Golgi in T cells. As an alternative possibility, Golgi-associated ATG16L may play autophagy-independent functions in these cells, as recently reported in Dictyostelium discoideum (Karow et al, 2020).…”

ATG16L1 functions in cell homeostasis beyond autophagy

“…The interaction between the WDD and the V-ATPase observed in the context of an infection and discussed in ATG16L1's WDD recruits the LC3conjugation machinery to lipidate PE at specific single membranes might be implicated in the mechanism by which ATG5 and ATG16L1 regulate exosome maturation and release [70,77] Finally, recent work in the ameba D. discoideum not only confirmed the essential function of the ATG12-ATG5/ATG16L1 complex in canonical autophagy, but also supported an autophagy-independent function of the complex and its individual components in the regulation of phagocytosis and pinocytosis. The data indicate that engagement of the complex in endocytic events might have evolved early [109].…”