The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions

Abstract

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome.

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

“…During growth in nutrient‐rich media, many of the D. discoideum autophagy genes appear to be dispensable (Otto et al, , ). Known exceptions to this are atg8a and atg9 , where their loss leads to reduced growth (Tung et al, ; Messling et al, ). Atg9 is found on distinct Golgi‐derived vesicles that are a source of autophagosomal membrane (they fuse to the autophagosomal outer membrane) and it is consequently involved in forming autophagosomes (Orsi et al, ; Yamamoto et al, ).…”

“…This signalling mechanism may also initiate developmental autophagy in D. discoideum (Otto et al, ). The independent loss of atg5 , atg6 , atg7 , atg8a , atg9 or atg101 leads to multi‐tipped aggregates (Otto et al, ; Calvo‐Garrido et al, ; Tung et al, ; Messling et al, ). Multi‐tipped aggregates are a characteristic phenotype of D. discoideum autophagy mutants, and this has been used to identify other autophagy‐regulating proteins in D. discoideum multiple tip mutations (e.g.…”

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).…”

“…D. discoideum harbours two paralogues, ATG8a and ATG8b, which have distinct functions in canonical autophagy. It was shown that they associate to the autophagosome in succession and that ATG8b mainly localises at the outer membrane of the autophagosome while ATG8a is mainly present at the inner membrane (Matthias et al, 2016, Messling et al, 2017. This differential localisation supports a more prominent role for ATG8b as an adapter for the autophagy machinery and in autophagosome lysosome fusion while ATG8a appears to function mainly as a binding partner for autophagy receptors.…”

“…This differential localisation supports a more prominent role for ATG8b as an adapter for the autophagy machinery and in autophagosome lysosome fusion while ATG8a appears to function mainly as a binding partner for autophagy receptors. On the basis of function and localisation it was inferred that ATG8b is likely the D. discoideum orthologue of the GABARAP and ATG8a of the LC3 subfamily in mammals (Table 1) (Messling et al, 2017). The autophagosomal membrane further expands through the incorporation of membrane lipids, engulfs entire organelles or parts of the cytoplasm and finally closes into a double-membrane structure, the autophagosome (Lamb et al, 2013, Stanley et al, 2014.…”