The two Dictyostelium autophagy eight proteins, ATG8a and ATG8b, associate with the autophagosome in succession

Abstract

Autophagy is an ancient cellular pathway that is conserved from yeast to man. It contributes to many physiological and pathological processes and plays a major role in the degradation of proteins and/or organelles in response to starvation and stress. In the autophagic process cytosolic material is captured into double membrane-bound vesicles, the autophagosomes. After fusion with lysosomes, the cargo is degraded in the generated autolysosomes and then recycled for further use. Autophagy 8 (ATG8, in mammals LC3), a well-established marker of autophagy, is covalently linked to phosphatidylethanolamine on the autophagic membrane during autophagosome formation. Bioinformatic analysis of the Dictyostelium genome revealed two atg8 genes which encode the ATG8a and ATG8b paralogs. They are with around 14kDa similar in size, 54 % identical to one another and more closely related to the corresponding proteins in fungi and plants than in animals. For ATG8a we found a strong up-regulation throughout the 24h developmental time course while ATG8b expression was highest in vegetative cells followed by a moderate reduction during early development. Confocal microscopy of fluorescently tagged ATG8a and ATG8b in vegetative AX2 wild-type and in ATG9(-) cells showed that both proteins mainly co-localized on vesicular structures with a diameter above 500nm while those smaller than 500nm were predominantly positive for ATG8b. In ATG9(-) cells we found a strong increase in the relative abundance of ATG8a-positive large vesicular structures and of total ATG8b-positive structures per cell indicating autophagic flux problems in this mutant. We also found that vesicular structures positive for ATG8a and/or ATG8b were also positive for ubiquitin. Live cell imaging of AX2 and ATG9(-) cells co-expressing combinations of red and green tagged ATG8a, ATG8b or ATG9 revealed transient co localizations of these proteins. Our results suggest that ATG8b associates with nascent autophagosomes before ATG8a. We further find that the process of autophagosome formation in Dictyostelium is highly dynamic. We infer from our data that Dictyostelium ATG8a and ATG8b have distinct functions in autophagosome formation and that ATG8b is the functional orthologue of the mammalian LC3 subfamily and ATG8a of the GABARAP subfamily.

Independent losses and duplications of autophagy‐related genes in fungal tree of life

“…The three subfamilies of animal Atg8s differ in their subcellular localization, expression profiles and functions in the autophagy process (Shpilka et al, 2011). Dictyostelium discoideum also has two ATG8 genes that have different expression profiles and functions (Matthias et al, 2016). In T. rubrum, the two ATG8 genes also differed in the expression profiles, suggesting that they may also have distinct roles in the autophagy process.…”

Autophagy inDictyostelium: Mechanisms, regulation and disease in a simple biomedical model

“…31 D. discoideum possess 2 Atg8 paralogs (Atg8a and b), which are both good markers of autophagosomes although they play partially nonredundant roles (discussed below). 32 Another useful marker is Atg18, the ortholog of mammalian WIPI2. Atg18 localizes to omegasomeanchored phagophores (the precursors to autophagosomes) at initiation events but, in contrast to Atg8, dissociates immediately after autophagosome formation is completed.…”

“…Immunolabeling of endogenous autophagy proteins allows a variety of markers to be observed, and several good antibodies against D. discoideum Atg8 have been generated. 32,35 While these antibodies are useful for many experiments, fixed samples lack dynamic information. It is therefore often useful to perform live-cell imaging and track markers in real-time to dissect the different phases of autophagosome formation.…”

“…Live cell imaging of D. discoideum cells expressing RFP-Atg8a and GFP-Atg8b showed that Atg8b associates with autophagosomes before ATG8a. 32 In mammals, LC3 subfamily members are involved in the elongation of the phagophore, whereas the GABARAP subfamily seem to be essential for a later stage in maturation. 71,72 Thus, Atg8b is likely the functional ortholog of the mammalian LC3 subfamily, which is also supported by the grouping in the evolutionary tree (Fig.…”

Dictyostelium discoideum and autophagy – a perfect pair

“…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.…”