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

Abstract

Autophagy is a highly conserved intracellular degradative pathway that is crucial for cellular homeostasis. During autophagy, the core autophagy protein ATG12 plays, together with ATG5 and ATG16, an essential role in the expansion of the autophagosomal membrane. In this study we analyzed gene replacement mutants of atg12 in Dictyostelium discoideum AX2 wild-type and ATG16‾ cells. RNAseq analysis revealed a strong enrichment of, firstly, autophagy genes among the up-regulated genes and, secondly, genes implicated in cell motility and phagocytosis among the down-regulated genes in 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 ATG12 and ATG16 act as a functional 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 independent of each other. In summary, we show that ATG12 and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy.

Dictyostelium discoideum and autophagy – a perfect pair

“…As a consequence, autophagy malfunction generally results in reduced cell survival upon nitrogen starvation and in developmental abnormalities in the affected strains, which also supports the identification of autophagy-deficient mutants in the laboratory (Fischer et al, 2019, King et al, 2013, Mesquita et al, 2017, Otto et al, 2003. The developmental phenotypes range from a complete lack of aggregation to the formation of extremely small and crippled fruiting bodies with drastically reduced spore viability (Calvo-Garrido et al, 2010, Fischer et al, 2019, Mesquita et al, 2015, Messling et al, 2017, Otto et al, 2003, Otto et al, 2004, Tung et al, 2010, Xiong et al, 2015. Differences in the importance of the respective autophagy proteins for the functioning of autophagy and additional non-autophagic functions for some of these proteins are likely responsible for the diverse observed phenotypes (Fischer et al, 2019).…”

“…Moreover, the unconventional secretion of AcbA, the precursor of the signalling peptide SDF-2 (spore differentiation factor 2), is indispensable for spore formation and depends on autophagy (Duran et al, 2010). As a consequence, autophagy malfunction generally results in reduced cell survival upon nitrogen starvation and in developmental abnormalities in the affected strains, which also supports the identification of autophagy-deficient mutants in the laboratory (Fischer et al, 2019, King et al, 2013, Mesquita et al, 2017, Otto et al, 2003. The developmental phenotypes range from a complete lack of aggregation to the formation of extremely small and crippled fruiting bodies with drastically reduced spore viability (Calvo-Garrido et al, 2010, Fischer et al, 2019, Mesquita et al, 2015, Messling et al, 2017, Otto et al, 2003, Otto et al, 2004, Tung et al, 2010, Xiong et al, 2015.…”

“…Loss of any of the components of this complex led to a reduction or even abolishment of LAP function in mouse embryonic fibroblasts (MEFs) and mouse bone marrow-derived macrophages (Huang et al, 2009, Kageyama et al, 2011, Lai and Devenish, 2012. Investigation of the phagocytic and macropinocytic activity of the D. discoideum ATG9‾, ATG16‾, ATG9‾/16‾, ATG12‾ and ATG12‾/16‾ strains showed that the encoded proteins have functions in phagocytosis of bacteria and yeast and also in the uptake of nutrients via macropinocytosis (Fischer et al, 2019, Tung et al, 2010, Xiong et al, 2015. However, this is only circumstantial evidence and thus far LAP has not been directly demonstrated in D. discoideum.…”

“…The developmental phenotypes range from a complete lack of aggregation to the formation of extremely small and crippled fruiting bodies with drastically reduced spore viability (Calvo-Garrido et al, 2010, Fischer et al, 2019, Mesquita et al, 2015, Messling et al, 2017, Otto et al, 2003, Otto et al, 2004, Tung et al, 2010, Xiong et al, 2015. Differences in the importance of the respective autophagy proteins for the functioning of autophagy and additional non-autophagic functions for some of these proteins are likely responsible for the diverse observed phenotypes (Fischer et al, 2019). Indeed, in recent years more and more non-autophagic functions of core autophagy proteins have been reported , Mauthe et al, 2016, Nam et al, 2017, Xiong et al, 2015.…”

Dictyostelium as model for studying ubiquitination and deubiquitination

“…In an analogous fashion to classic ubiquitination, the ubiquitin-like protein ATG12 is transferred from the E1-like enzyme ATG7 via the E2-like protein ATG10 to ATG5, and the ATG12~5 conjugate is formed. In Dictyostelium the characterization of the ATG12 and/or ATG16 null mutants has highlighted the role of these proteins in autophagy-independent process as macropinocytosis, protein homeostasis, fruiting body formation, in addition to their role in canonical autophagy (Xiong et al, 2015;Fischer et al, 2019).…”