ATG8/LC3-mediated autophagosome formation is a key rate-limiting step in the process of autophagy. The parasitic protist Toxoplasma gondii possesses a single ATG8 homolog (TgATG8), which can localize to either cytosolic autophagosome involved in delivery of autophagic material in bradyzoites, or the outermost membrane of apicoplast, a nonphotosynthetic plastid-like organelle, responsible for maintaining homeostasis in tachyzoites. However, mechanisms that regulate TgATG8 remain insufficiently understood. Here, a TgATG7 conditional knockdown line that we have generated is severely impaired in parasite's growth and exhibits significant defects in the organelle level, strikingly with a fragmentation of the mitochondrial network and a loss of the apicoplast. Specific TgATG7C1133S point mutant complemented line showed that these defects were associated with its E1-type enzyme activity. Both depletion of TgATG7 and mutation of its catalytic cysteine 1133 hindered TgATG8 lipidation and apicoplast localization. Unexpectedly, we also found that depletion of TgATG7 reduced the unlipidated TgATG8 protein level. Subsequently, we determined that TgATG7 was able to interact with TgATG8 directly via its C-terminal domain and multi-monoubiquitination stimulated proteasome-dependent degradation of TgATG8, while TgATG7 could inhibit the degradation through stabilization of TgATG8. Additionally, we identified a putative TgATG8 interacting fragment of TgATG7, 1281-1290aa. Depletion of the fragment impaired the parasite growth and apicoplast inheritance. To our knowledge, our study is the first to elucidate the role of TgATG7 and the ubiquitin-proteasome system in synergistically regulating the non-lipidated pool of TgATG8, suggesting a potential homeostatic mechanism responsible for balancing autophagic activity in T. gondii.
Keywords: ATG7; ATG8; Apicoplast; Autophagy; Toxoplasma gondii; Ubiquitination.
Copyright © 2023. Published by Elsevier B.V.