Autophagy-Related Protein ATG8 Has a Noncanonical Function for Apicoplast Inheritance in Toxoplasma gondii

Abstract

Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. This mechanism involves the formation of a double-membrane structure called the autophagosome that sequesters cellular components to be degraded. To orchestrate this process, yeasts and animals rely on a conserved set of autophagy-related proteins (ATGs). Key among these factors is ATG8, a cytoplasmic protein that is recruited to nascent autophagosomal membranes upon the induction of autophagy. Toxoplasma gondii is a potentially harmful human pathogen in which only a subset of ATGs appears to be present. Although this eukaryotic parasite seems able to generate autophagosomes upon stresses such as nutrient starvation, the full functionality and biological relevance of a canonical autophagy pathway are as yet unclear. Intriguingly, in T. gondii, ATG8 localizes to the apicoplast under normal intracellular growth conditions. The apicoplast is a nonphotosynthetic plastid enclosed by four membranes resulting from a secondary endosymbiosis. Using superresolution microscopy and biochemical techniques, we show that TgATG8 localizes to the outermost membrane of this organelle. We investigated the unusual function of TgATG8 at the apicoplast by generating a conditional knockdown mutant. Depletion of TgATG8 led to rapid loss of the organelle and subsequent intracellular replication defects, indicating that the protein is essential for maintaining apicoplast homeostasis and thus for survival of the tachyzoite stage. More precisely, loss of TgATG8 led to abnormal segregation of the apicoplast into the progeny because of a loss of physical interactions of the organelle with the centrosomes.

TgDrpC, an atypical dynamin‐related protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division

“…Immunofluorescence images showed that the ATrx1 signal is disrupted; moreover, no apicoplast DNA signal is detectable by DAPI in those parasites (Fig. C), a phenotype consistent with loss of the apicoplast as previously reported by Lévêque et al (Leveque et al , ). A similar pattern is observed with the Golgi in TgDrpC‐HA‐DD parasites grown without Shld1: 85 ± 7% of swollen vacuoles contained parasites in which the Golgi appeared as dispersed puncta in contrast to a solid structure typical of normal parasites (Fig.…”

Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection

“…Pru strains expressing tdTomato-ATG8 or a tdTomato-ATG8 G124A lipidation mutant were generated by modifying the pTub-tdTomato-TgATG8-CAT plasmid described in Leveque et al 13 via replacement of CAT with BLE and cloning in a larger tubulin promoter fragment encompassing -2730 to -1 relative to the tubulin start codon. Plasmids were linearized with Nar I at -1318 for single crossover integration into the tubulin locus.…”

“…Toxoplasma and related apicomplexans including malaria parasites express a limited repertoire of autophagy (ATG) proteins 12 . These parasites also unconventionally require certain ATG proteins for maintenance of the apicoplast 13, 14 , a remnant non-photosynthetic chloroplast. Autophagosome-like structures have been observed under nutrient limiting conditions 15, 16 , but the extent to which the parasite encounters such conditions during infection remains unknown.…”

Toxoplasma gondii autophagy-related protein ATG9 is crucial for the survival of parasites in their host

“…, ), where it plays a noncanonical role in organelle inheritance during cell division (Lévêque et al . , ). The apicoplast is involved in several key metabolic pathways, so it is essential for the survival of the parasites (Sheiner, Vaidya, & McFadden, ).…”